CN116744941A

CN116744941A - RNA production

Info

Publication number: CN116744941A
Application number: CN202180092586.7A
Authority: CN
Inventors: T·齐根哈尔斯; A·库恩; S·费塞尔
Original assignee: Biotechnology Europe Inc
Current assignee: Biotechnology Europe Inc
Priority date: 2020-12-09
Filing date: 2021-12-07
Publication date: 2023-09-12

Abstract

The present disclosure provides techniques for performing in vitro transcription that are capable of producing product RNA preparations with reduced levels of certain contaminants (e.g., abnormal products), particularly double-stranded RNAs (dsRNA).

Description

RNA production

Technical Field

The present invention relates to a method for reducing dsRNA during in vitro transcription by stepwise nucleotide addition. The invention also relates to nucleic acids produced by the methods of the invention and the use of such nucleic acids in methods of treating a subject in need thereof.

Background

In vitro production of RNA is becoming increasingly important in biotechnology and pharmaceutical industry. It is important and valuable to improve manufacturing processes, particularly those that can produce high quality RNAs (e.g., mRNA) on a large scale, including in particular therapeutic grade RNAs (e.g., mRNA).

Disclosure of Invention

In vitro transcribed RNAs (e.g., by T7 RNA polymerase (RNAP)) may contain abnormal products, including significant levels of abnormal products. Without wishing to be bound by theory, it is proposed that some or all of such products may be produced by the unusual activity of the RNAP utilized. One such abnormal product is dsRNA, which may prove particularly problematic, for example, in view of its propensity to induce inflammatory cytokines and/or activate immune effector proteins, which may in particular lead to inhibition of protein synthesis. dsRNA is often the primary contaminant of the RNA transcription reaction in vitro.

Typically, abnormal products, particularly dsRNA, are removed from an in vitro transcribed RNA preparation by purification; a variety of purification techniques are available (e.g., via LiCl and/or alcohol-based precipitation, size exclusion and/or ion exchange chromatography, silica matrix purification, ion-pair reversed-phase high performance liquid chromatography [ HPLC ], cellulose-based separations, etc.). However, most or all such purification strategies may be impractical and/or otherwise undesirable, particularly for commercial scale and/or pharmaceutical grade production, etc., particularly because they often remove the desired RNA product along with the abnormal product (and/or other contaminants), resulting in substantial loss of the undesired RNA product.

The present disclosure provides inter alia the insight that surprisingly limiting the amount of UTP or a functional analogue thereof when synthesizing RNA by a transcription reaction and supplementing the reaction mixture with UTP or a functional analogue thereof during the transcription reaction can yield RNA of increased purity, reduced immunogenicity and good stability.

Furthermore, the present disclosure provides insight that benefits may be obtained by reducing the production of abnormal products (and/or other contaminants) in the first place. The present disclosure provides techniques for performing in vitro transcription that can produce product RNA formulations with reduced levels of certain contaminants (e.g., abnormal products), particularly dsRNA. Advantages of the provided techniques include, but are not limited to, more efficient manufacturing, including higher product RNA yield (e.g., less product loss during processing), fewer processing steps (which may help reduce product loss), lower production costs, shorter production time lines, and the like. Furthermore, the present disclosure teaches that the improved production techniques (e.g., improved transcription reaction conditions) provided even have various advantages (including the aforementioned advantages) over improved purification techniques.

In one aspect, the present invention relates to a method of producing RNA, the method comprising transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP and/or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with a composition comprising UTP or a functional analog thereof and substantially no CTP or ATP or functional analog thereof during the transcription reaction.

In one aspect, the present invention relates to a method of producing RNA, the method comprising transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of CTP or a functional analog thereof is equal to the initial concentration of ATP or a functional analog thereof, and wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with UTP or a functional analog thereof during the transcription reaction.

In one aspect, the present invention relates to a method of producing a composition comprising RNA having reduced double stranded (ds) RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP and/or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with a composition comprising UTP or a functional analog thereof and substantially free of CTP or ATP or a functional analog thereof during the transcription reaction.

In one aspect, the present invention relates to a method of producing a composition comprising RNA having reduced double stranded (ds) RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of CTP or a functional analog thereof is equal to the initial concentration of ATP or a functional analog thereof, and wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with UTP or a functional analog thereof during a transcription reaction.

In some embodiments, the double stranded (ds) RNA content of the RNA-containing composition is reduced compared to the dsRNA content of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

In some embodiments, the composition comprising RNA has reduced immunogenicity as compared to the immunogenicity of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

In some embodiments, uridine Triphosphate (UTP) or a functional analog thereof is present at an initial concentration that limits transcription rate.

In some embodiments, the ratio of the starting concentration of Uridine Triphosphate (UTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

In some embodiments, the reaction mixture is replenished with Uridine Triphosphate (UTP) or a functional analog thereof when the concentration of UTP or a functional analog thereof approaches depletion.

In some embodiments, the reaction mixture is supplemented at least once during the transcription reaction with Uridine Triphosphate (UTP) or a functional analog thereof.

In some embodiments, the reaction mixture is continuously replenished with Uridine Triphosphate (UTP) or a functional analog thereof during the transcription reaction.

In some embodiments, the reaction mixture is periodically replenished during the transcription reaction with Uridine Triphosphate (UTP) or a functional analog thereof.

In some embodiments, the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analog thereof to maintain or restore an initial ratio of concentration of UTP or a functional analog thereof to concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

In some embodiments, the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analog thereof until the transcription reaction is complete.

In some embodiments, the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof is lower than the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof. In some embodiments, guanosine Triphosphate (GTP) or a functional analog thereof is preferably present at an initial concentration that limits the transcription rate.

In some embodiments, the ratio of the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

In some embodiments, the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

In some embodiments, when the concentration of Guanosine Triphosphate (GTP) or a functional analog thereof is near depletion, the reaction mixture is replenished with GTP or a functional analog thereof.

In some embodiments, the reaction mixture is supplemented at least once during the transcription reaction with Guanosine Triphosphate (GTP) or a functional analogue thereof.

In some embodiments, the reaction mixture is continuously replenished with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

In some embodiments, the reaction mixture is periodically replenished during the transcription reaction with Guanosine Triphosphate (GTP) or a functional analog thereof.

In some embodiments, the supplementation of the reaction mixture with Guanosine Triphosphate (GTP) or a functional analog thereof maintains or restores the initial ratio of the concentration of GTP or a functional analog thereof to the concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

In some embodiments, the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof until the transcription reaction is completed.

In some embodiments, the provided methods do not comprise supplementing the transcription mixture with Cytidine Triphosphate (CTP) and/or Adenosine Triphosphate (ATP) or functional analogs thereof during the transcription reaction.

In some embodiments, the reaction mixture comprises a starting nucleotide corresponding to a first nucleotide in the RNA molecule.

In some embodiments, the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

In some embodiments, the starting nucleotide is a 5 'cap or 5' cap analog.

In some embodiments, the 5 'cap or 5' cap analogue is selected from the group consisting of: g < 5']ppp[5']G、m7G[5']ppp[5']G、m ₃ ^2,2,7 G[5']ppp[5']G、m ₂ ^7,3'-O G[5']ppp[5']G(3'-ARCA)、m ₂ ^7,2'-O GpppG(2'-ARCA)、m ₂ ^7,2'-O Gpp _S pG D1(β-S-ARCA D1)、m ₂ ^7,2'-O Gpp _S pG D2 (. Beta. -S-ARCA D2) and m ₂ ^7,3'-O Gppp(m ₂ '-O)ApG(CC413)。

In some embodiments, the 5 'cap or 5' cap analogue in the reaction mixture is present in excess compared to Guanosine Triphosphate (GTP) or a functional analogue thereof.

In some embodiments, the ratio of the starting concentration of 5 'cap or 5' cap analogue to the starting concentration of Guanosine Triphosphate (GTP) or a functional analogue thereof is between about 2:1 and about 20:1.

In some embodiments, the ratio of the starting concentration of 5 'cap or 5' cap analogue to the starting concentration of Guanosine Triphosphate (GTP) or a functional analogue thereof is about 4:1.

In some embodiments, the reaction mixture further comprises an RNA polymerase, a buffer, and at least one monovalent or divalent cation.

In some embodiments, the cation is Li ⁺ 、Na ⁺ 、K ⁺ 、NH ⁴⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ 。

In some embodiments, the RNA polymerase is selected from the group consisting of: t7RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.

In some embodiments, the functional analog of Uridine Triphosphate (UTP) is selected from the group consisting of: pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP and 4-thio-UTP.

In some embodiments, the functional analog of Guanosine Triphosphate (GTP) is selected from the group consisting of: 7-deaza-GTP, N1-methyl-GTP and O6-methyl-GTP.

In some embodiments, the DNA template encodes one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

In some embodiments, the RNA comprises one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

In some embodiments, the RNA encodes at least one peptide or protein.

In some embodiments, the RNA is mRNA.

In some embodiments, the RNA is self-replicating RNA.

In some embodiments, the RNA produced by the methods of the invention has a length of 200 to 20000 nucleotides, 200 to 12000 nucleotides, 200 to 8000 nucleotides, 500 to 5000 nucleotides, 500 to 2500 nucleotides, particularly 600 to 2500 nucleotides or 800 to 2000 nucleotides.

In some embodiments, the pH of the reaction mixture remains substantially constant during the transcription reaction.

In some embodiments, the progress of the transcription reaction is monitored in real time.

In some embodiments, the method is performed using a bioreactor.

In one aspect, the invention relates to an RNA produced by the method of the invention.

In one aspect, the invention relates to a composition comprising RNA produced by the method of the invention.

In one aspect, the invention relates to a method of treating a subject, the method comprising the steps of: (i) Obtaining RNA produced by the methods of the invention, or obtaining a composition comprising RNA produced by the methods of the invention, and (ii) administering the RNA or the composition comprising RNA to a subject.

In one aspect, the invention relates to a method for producing RNA by in vitro transcription, wherein the method comprises limiting the concentration of UTP or a functional analogue thereof during an in vitro transcription reaction.

In one aspect, the invention relates to an in vitro transcription reaction comprising: an RNA template comprising a promoter that directs transcription of the template to produce a transcript optionally having polyA sequence elements; RNA polymerase acting on the promoter; and Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP) and Uridine Triphosphate (UTP) or functional analogues thereof, wherein the initial concentration of UTP or functional analogue thereof is lower than the concentration of CTP and/or ATP or functional analogue thereof.

In one aspect, the invention relates to a method of treating a subject by administering to the subject an RNA produced by the method of the invention or a composition comprising an RNA produced by the method of the invention.

In one aspect, the present invention relates to a method for reducing the amount of double stranded RNA produced by in vitro transcription of RNA, the method comprising in vitro transcribing RNA from a template under transcription conditions wherein the amount or concentration of UTP or a functional analogue thereof is a limited amount or concentration of RNA transcription compared to the amount or concentration of one or more of ATP, CTP and/or GTP or a corresponding analogue thereof.

In one aspect, the present invention relates to a method for reducing the amount of double stranded RNA in a composition comprising RNA produced by in vitro transcription of RNA, the method comprising in vitro transcribing RNA from a template under transcription conditions wherein the amount or concentration of UTP or a functional analogue thereof is a limited amount or concentration of RNA transcription compared to the amount or concentration of one or more of ATP, CTP and/or GTP or a corresponding analogue thereof.

In one aspect, the present invention relates to a method for reducing the immunogenicity of a composition comprising RNA produced by in vitro transcription of RNA, the method comprising in vitro transcribing RNA from a template under transcription conditions wherein the amount or concentration of UTP or a functional analogue thereof is a limited amount or concentration of RNA transcription compared to the amount or concentration of one or more of ATP, CTP and/or GTP or a corresponding analogue thereof.

In one embodiment, the double stranded RNA is the result of annealing at least two different RNA molecules to each other, i.e., the result of intermolecular binding. In one embodiment, the double stranded RNA is the result of intramolecular binding, i.e., the portion of the RNA molecule anneals to itself, e.g., in the case of a transcript that is reverse folded to itself.

Drawings

Fig. 1 shows exemplary results of dsRNA content generated by an IVT reaction that transcribes unmodified RNA with stepwise addition of NTP. RNA was transcribed in vitro with a reduced starting concentration (restriction) of the indicated NTPs. G-GTP, A-ATP, U-UTP, A/U-ATP+UTP. The limited NTP is fed stepwise into the IVT reaction until all NTPs reach the final concentration of NTP. All RNAs were co-transcribed capped using the CC413 cap analogue. Restriction of GTP was used as a control. A. According to the type of NTP fed during the reaction, the RNA yield was unaffected compared to the control. B. RNA integrity was unaffected compared to the control, according to the NTP type fed during the reaction. C. When ATP was fed, the dsRNA content was increased compared to the control, and when UTP was fed, the content was decreased. Both feed ATP and UTP eliminate each other's effects, resulting in dsRNA content comparable to the control (feed GTP). D. Capping efficiency was reduced when GTP was not fed compared to the control.

Fig. 2 shows exemplary results of dsRNA content generated by IVT reaction of unmodified RNA (decreasing capping efficiency) with stepwise addition of UTP or GTP and UTP. RNA was transcribed in vitro with a reduced starting concentration (restriction) of the indicated NTPs. G-GTP, U-UTP, G/U-GTP+UTP. The limited NTP is fed stepwise into the IVT reaction until all NTPs reach the final concentration of NTP. Restriction of GTP was used as a control. All RNAs were co-transcribed capped using the D1- β -S1 ARCA cap analogue. A. When UTP or UTP and GTP are fed in combination under these reaction conditions, the RNA yield increases compared to the control. B. When UTP or GTP and UTP are fed, the integrity of the purified RNA is reduced. When GTP and UTP are fed in combination, RNA integrity is rescued to a level when the IVT reaction is GTP feed (control). C. dsRNA content was reduced by feeding UTP compared to control GTP feed. Feeding both GTP and UTP also reduced dsRNA content, but to a lesser extent than feeding UTP alone. D. Capping efficiency was reduced when UTP was fed alone compared to the control, but was rescued by combining GTP and UTP.

FIG. 3 shows exemplary results of dsRNA content generated by IVT reactions transcribing RNA containing m1 ψTP with stepwise addition of N1-methyl pseudouridine (m 1 ψTP) or m1 ψTP and GTP. RNA was transcribed in vitro with a reduced starting concentration (restriction) of the indicated NTPs. G-GTP, m1 ψ -m1 ψ TP, G/m1 ψ -GTP+m1 ψ TP. The limited NTP is fed stepwise into the IVT reaction until all NTPs reach the final concentration of NTP. Restriction of GTP was used as a control. All RNAs were co-transcribed capped using CC413 GAG cap analogues. A. According to the type of NTP fed during the reaction, the RNA yield was unaffected compared to the control. B. When m1 ψtp was fed, RNA integrity was reduced compared to the control. When fed GTP and m1YTP are combined, RNA integrity is rescued to the level of the GTP feed control. C. dsRNA content was reduced by feeding m1 ψtp compared to standard GTP feed control. Feeding GTP and m1 ψtp reduced dsRNA content, comparable to feeding m1 ψtp alone. D. Capping efficiency was reduced when feeding m1 ψtp compared to the control, but was rescued by feeding GTP and m1 ψtp.

Fig. 4 shows exemplary results of higher order structures of RNA products obtained using circular dichroism.

Certain definitions

Certain terms as used herein may be understood as "A multilingual glossary of biotechnological terms (IUPAC Recommendations)", H.G.W.Leuenberger, B.Nagel and H.Edit Helvetica Chimica Acta, CH-4010Basel, switzerland, (1995).

Moreover, an indication of the relative amounts of components characterized by a generic term generally refers to the total amount of all specific variants or members encompassed by the generic term. If a component defined by a generic term is specified to be present in a certain relative amount, and if the component is further characterized as a particular variant or member encompassed by the generic term, then that means that no other variant or member encompassed by the generic term is additionally present, so that the total relative amount of the components encompassed by the generic term exceeds the specified relative amount; more preferably, there are no other variants or members at all encompassed by the generic term.

A: the use of the terms "a/an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

About or about: the term "about" or "approximately" as used herein to refer to a value refers to a value that is similar in context to the stated reference value. Generally, those skilled in the art who are familiar with the context will understand the relative degree of variation involved in "about" or "approximately" in this context. Those of skill in the art will understand that in many embodiments (as will be understood from the context), the term "about" means about or near, and in the context of a value or range set forth herein, preferably means +/-10% of the value or range. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the referenced values.

And (3) application: as used herein, the term "administering" generally refers to administering a composition to a subject or system. Those of ordinary skill in the art will appreciate a variety of routes that may be used for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or include, for example, one or more of dermal topical, intradermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, the administration may be intramuscular. In some embodiments, administration may involve administration as intermittent (e.g., multiple doses separated in time) and/or periodic (e.g., individual doses separated by the same period of time) administrations. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time.

The preparation method comprises the following steps: generally, the term "agent" as used herein is used to refer to an entity (e.g., a lipid, a metal, a nucleic acid, a polypeptide, a polysaccharide, a small molecule, etc., or a complex, combination, mixture, or system thereof [ e.g., a cell, tissue, organism ]) or phenomenon (e.g., heat, electrical current or electric field, magnetic force or magnetic field, etc.). Where appropriate, the term may be used to refer to or comprise an entity of a cell or organism or a fraction, extract or component thereof, as will be clear to a person skilled in the art from the context. Alternatively or additionally, as the context will be clear, the term may be used to refer to natural products found in and/or obtained from nature. In some cases, as will also be clear from the context, the term may be used to refer to one or more artificial entities, as it is designed, engineered, and/or created by the action of a person's hand, and/or not found in nature. In some embodiments, the agent may be used in isolated or pure form; in some embodiments, the agent may be used in crude form. In some embodiments, the potential agents may be provided as collections or libraries that may be screened, for example, to identify or characterize the active agents therein. In some cases, the term "agent" may refer to or comprise a compound or entity of a polymer; in some cases, the term may refer to a compound or entity that includes one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

An analog: as used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or portions with the reference substance. In general, "analogs" exhibit significant structural similarity to a reference substance, e.g., sharing a core or consistent structure, but also differ in some discrete ways. In some embodiments, the analog is a substance that can be generated from a reference substance, for example, by chemically manipulating the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process that is substantially similar (e.g., shares multiple steps with) a synthetic process that generates a reference substance. In some embodiments, the analog is or may be generated by performing a different synthetic process than that used to generate the reference substance.

Antibody preparation: as used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes an immunoglobulin structural element sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, an antibody agent may include one or more humanized, primatized, chimeric, etc., sequence elements, as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, the antibody agent used according to the present disclosure is in a form selected from, but not limited to: intact IgA, igG, igE or IgM antibodies; bispecific or multispecific antibodies (e.g., Etc.); antibody fragments, such as Fab fragments, fab ' fragments, F (ab ') 2 fragments, fd ' fragments, fd fragments, and isolated Complementarity Determining Regions (CDRs), or a collection thereof; a single chain Fv; a polypeptide-Fc fusion; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); camelidae antibodies; masking antibodies (e.g.)>) The method comprises the steps of carrying out a first treatment on the surface of the Small modular immunopharmaceuticals ("SMIPsTM"); single-chain or tandem diabodies->VHH；/> A minibody; />Ankyrin repeat protein or-> DART; TCR-like antibodies; /> A micro protein; />And +.>In some embodiments, the antibody may lack covalent modifications (e.g., attachment of glycans) that are present when naturally occurring. In some embodiments, antibodies can contain covalent modifications (e.g., glycans, payloads [ e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.)]Or other side groups [ e.g., polyethylene glycol, etc. ]]Is attached). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as Complementarity Determining Regions (CDRs); in some embodiments, the antibody agent is or comprises at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) whose amino acid sequence comprises substantially the same as a CDR found in a reference antibody CDR). In some embodiments, the CDR included is substantially identical to the reference CDR in that it is identical in sequence or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that it shows at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is deleted, added, or substituted as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.

The skilled artisan can prepare antibody agents using methods known in the art and commercially available services and kits. For example, methods of preparing monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Other antibodies suitable for use in the present disclosure are described, for example, in the following publications: antibodies A Laboratory Manual, second edition, edward A. Greenfield. Cold Spring Harbor Laboratory Press (2013, 9, 30); making and Using Antibodies: a Practical Handbook, second edition, editor Gary c.howard and Matthew r.kaser.crc Press (2013, 7, 29); antibody Engineering: methods and Protocols, second edition (Methods in Molecular Biology) Patrick chames. Humana Press (2012, 8, 21); monoclonal Antibodies: methods and Protocols (Methods in Molecular Biology) editors Vincent oscipow and Nicolas fischer humana Press (2014, 2, 12); and Human Monoclonal Antibodies: methods and Protocols (Methods in Molecular Biology) Michael Steinitz. Humana Press (30, 9, 2013)).

Antibodies can be produced by standard techniques, for example by immunization with the appropriate polypeptide or one or more portions thereof, or by use of phage display libraries. If polyclonal antibodies are desired, the selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing one or more desired epitopes, optionally hapten to another polypeptide. Depending on the host species, various adjuvants may be used to increase the immune response. Such adjuvants include, but are not limited to, freund's adjuvant, mineral gels (such as aluminum hydroxide) and surface active substances (such as lysolecithin), pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from immunized animals is collected and treated according to known procedures. If the serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies may be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are well known in the art.

Antigen: the term "antigen" as used herein refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, the antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, the antigen triggers a cellular response (e.g., involves T cells whose receptor specifically interacts with the antigen). In some embodiments, the antigen binds to an antibody and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biopolymer [ e.g., other than a nucleic acid or amino acid polymer), and the like. In some embodiments, the antigen is or comprises a polypeptide. In some embodiments, the antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that in general, the antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., along with other materials, e.g., in extracts such as cell extracts or other relatively crude preparations containing sources of antigen). In some embodiments, the antigen used according to the invention is provided in crude form. In some embodiments, the antigen is a recombinant antigen.

And (3) autologous: the term "autologous" is used to describe anything derived from the same subject. For example, "autologous cells" refer to cells derived from the same subject. The introduction of autologous cells into a subject is advantageous because these cells overcome the immunological barrier that would otherwise lead to rejection reactions.

Allograft: the term "allograft" is used to describe anything that originates from a different individual of the same species. When the genes at one or more loci are different, two or more individuals are said to be allogeneic to each other.

Base pairing: as understood in the art, a "base pair" is a structural motif of a secondary structure in which two nucleotide bases associate with each other through hydrogen bonding between donor and acceptor sites on the bases. Complementary bases A, U and G, C are understood to be capable of forming stable base pairs by hydrogen bonding between donor and acceptor sites on the bases; the A, U and G, C base pairs are referred to as Watson-Crick base pairs. Weaker base pairs (called wobble base pairs) are formed by bases G and U (G: U). Base pairs A, U and G, C may be referred to as "canonical" base pairs. Other base pairs such as G: U (which is often found in RNA) and other relatively unusual base pairs (e.g., A: C; U: U) may be referred to as non-canonical base pairs.

Batch wise: as used herein, the term "batch" or "batch reaction" or similar terms refer to at least one discrete replenishment event of at least one component, (e.g., in some embodiments) specifically UTP or analog thereof, optionally at least one discrete replenishment event of other components (optionally multiple components replenished in the same discrete replenishment event).

Combining: it should be understood that the term "binding" as used herein generally refers to non-covalent association between or among two or more entities. "direct" bonding involves physical contact between entities or parts; indirect bonding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of situations, including studying the interacting entity or moiety alone or in the context of a more complex system (e.g., when covalently associated or otherwise associated with a carrier entity and/or in a biological system or cell).

A bioreactor: the term "bioreactor" as used herein refers to a vessel for in vitro transcription as described herein. The bioreactor may be of any size as long as it can be used for in vitro transcription. For example, in some embodiments, the bioreactor may be at least 0.5 liters, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters, or more, or any volume therebetween. The internal conditions of the bioreactor (including but not limited to pH and temperature) are typically controlled during in vitro transcription. The bioreactor may be constructed of any material (including glass, plastic or metal) suitable for in vitro transcription under the conditions as described herein. One of ordinary skill in the art will appreciate and be able to select an appropriate bioreactor volume for performing in vitro transcription.

Cap: as used herein, the term "cap" refers to or consists essentially of a nucleoside-5 ' -triphosphate comprising a 5' -end that is typically bound to uncapped RNA (e.g., uncapped RNA with 5' -diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, including for example, but not limited to, an N7-methylguanosine cap having a structure designated "m 7G". In some embodiments, the cap is or comprises a synthetic cap analogue similar to an RNA cap structure and having the ability to stabilize RNA if attached thereto, including for example, but not limited to, anti-reverse cap analogues (ARCA) known in the art. Those skilled in the art will appreciate that methods for binding caps to the 5' end of RNA are known in the art. For example, in some embodiments, the capped RNA can be obtained by in vitro capping of RNA having a 5 'triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system, including, for example, but not limited to, a vaccinia capping enzyme system or a saccharomyces cerevisiae (Saccharomyces cerevisiae) capping enzyme system. Alternatively, the capped RNA can be obtained by In Vitro Transcription (IVT) of a DNA template, wherein the IVT system contains cap analogues in addition to GTP, e.g. as known in the art. Non-limiting examples of Cap analogs include m7GpppG Cap analogs or N7-methyl-, 2 '-O-methyl-GpppG ARCA Cap analogs or N7-methyl-, 3' -O-methyl-GpppG ARCA Cap analogs or any commercially available Cap analogs including, for example, cleanCap (Trilink), ezcap, etc. In some embodiments, the cap analogue is or comprises a trinucleotide cap analogue. Various cap analogs are described herein and are known in the art, e.g., commercially available.

Codons: as understood in the art, the term "codon" refers to a base triplet in a coding nucleic acid that specifies which amino acid will be added next during protein synthesis at the ribosome.

The method is equivalent to that of: as used herein, the term "comparable" refers to two or more agents, entities, conditions, sets of conditions, etc., that may be different from each other but sufficiently similar to allow comparison between them, such that one of ordinary skill in the art will understand that a conclusion can be reasonably drawn based on the observed differences or similarities. In some embodiments, a comparable set of conditions, environment, individual or population is characterized by a plurality of substantially identical features and one or a small number of different features. Those of ordinary skill in the art will understand how the degree of consistency required for two or more such agents, entities, situations, condition sets in any given instance is considered comparable. For example, one of ordinary skill in the art will understand that when characterized by a sufficient number and type of substantially identical features, sets of conditions, individuals, or populations are comparable to one another to ensure a reasonable conclusion that the differences in the results or observed phenomena obtained under or using different sets of conditions, individuals, or populations are caused by or indicative of changes in those changed features.

Complementary: as used herein, the term "complementary" is used to refer to hybridization of oligonucleotides related by the base pairing rules. For example, the sequence "C-A-G-T" is complementary to the sequence "G-T-C-A". Complementarity may be partial or complete. Thus, any degree of partial complementarity is intended to be included within the scope of the term "complementarity" so long as the partial complementarity allows hybridization of the oligonucleotides. Partial complementarity is where one or more nucleic acid bases are mismatched according to the base pairing rules. Full or complete complementarity between nucleic acids is where each and every nucleic acid base matches another base under the base pairing rules. As understood in the art, percent complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "perfect complementarity" or "complete complementarity" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. In many embodiments, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. In certain embodiments, the degree of complementarity according to the present invention is 100%.

The method comprises the following steps: the term "comprising" is used in the context of this document to mean that other members may optionally be present in addition to the members of the list introduced by "comprising" unless explicitly specified otherwise. However, as a specific embodiment of the present invention, the term "comprising" is considered to cover the possibility that no further members are present, i.e. for the purpose of this embodiment "comprising" is to be understood to have the meaning of "consisting of … …".

Reduction, decrease, inhibition: as understood in the art, terms such as "reduce," "reduce," or "inhibit" may be used herein to refer to an overall and/or relatively reduced ability to cause, for example, an entity, event, frequency, activity, etc. In some embodiments, the decrease may be, for example, 5% or greater, 10% or greater, 20% or greater, 25% or greater, 30% or greater, 35% or greater, 40% or greater, 45% or greater, in some embodiments 50% or greater, 60% or greater, 70% or greater, and in some embodiments 75% or greater. In some embodiments, the decrease may be 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 7-fold or greater, 8-fold or greater, 9-fold or greater, 10-fold or greater, 15-fold or greater, 20-fold or greater, 25-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold or greater, 100-fold or greater, etc. In some embodiments, the decrease is assessed relative to an appropriate reference. In some embodiments, inhibition may be complete or "substantially complete," e.g., to an undetectable level, e.g., to zero or substantially zero.

Derivatives: as used herein, the term "derivative" refers to a structural analogue of a reference substance. That is, a "derivative" is a substance that exhibits significant structural similarity (e.g., shared core or shared structure) with a reference substance, but also differs in some discrete manner. In some embodiments, the derivative is a substance that can be generated from a reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be produced by performing a synthetic process that is substantially similar (e.g., sharing multiple steps) to a synthetic process that produces a reference substance. For example, in some embodiments, a "derivative" of a nucleic acid residue may be or comprise a difference in nucleotide base, sugar, or phosphate. In some embodiments, a "derivative" of a nucleic acid may be a nucleic acid that contains one or more non-naturally occurring nucleotides and/or nucleotide analogs. In some embodiments, the derivative of the nucleic acid is more stable than a comparable nucleic acid lacking the associated derivatization. In some embodiments, the term "derivative" is used to refer to a nucleic acid sequence that is a variant relative to a particular reference sequence; in some such embodiments, such derived (i.e., variant) sequences exhibit comparable or improved stability and/or translational efficiency relative to their parent reference sequence, e.g., when they replace such parent reference sequence in an RNA molecule.

And (3) detection: the term "detecting" is used broadly herein to include an appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, "detecting" may include determining, measuring, assessing, or analyzing the presence or absence, level, quantity, and/or location of an entity of interest. Including quantitative and qualitative determinations, measurements or evaluations, including semi-quantitative. Such determination, measurement or evaluation may be relative, for example when the entity of interest is detected relative to a control reference, or absolute. Thus, the term "quantization" when used in the context of quantizing an entity of interest may refer to absolute or relative quantization. Absolute quantification may be achieved by correlating the detection level of the entity of interest with a known control standard (e.g., by generating a standard curve). Alternatively, relative quantization may be achieved by comparing detection levels or amounts between two or more different entities of interest to provide a relative quantization for each of the two or more different entities of interest (i.e., relative to each other).

And (3) determining: those of ordinary skill in the art who review this description will understand that the step of "determining" may be accomplished using any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein, or by using any of these techniques. In some embodiments, manipulation involving a physical sample is determined. In some embodiments, the determination involves consideration and/or manipulation of data or information, for example, using a computer or other processing unit adapted to perform correlation analysis. In some embodiments, determining involves receiving relevant information and/or substances from a source. In some embodiments, the determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

Dosage form or unit dosage form: those skilled in the art will appreciate that the term "dosage form" may be used to refer to a physically discrete unit of active agent (e.g., therapeutic agent or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of active agent. In some embodiments, the amount is a unit dose amount (or whole portion thereof) suitable for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to the relevant population (i.e., using a therapeutic dosing regimen). It will be appreciated by those of ordinary skill in the art that the total amount of therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

Encapsulation: the term "encapsulate" is used herein to mean that at least a portion of a component is enclosed or surrounded by another material or another component in the composition. In some embodiments, the component may be completely enclosed or surrounded by another material or another component in the composition.

Excipient: as used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired property or effect (e.g., a desired consistency, delivery and/or stabilizing effect, etc.). In some embodiments, the term "excipient" is intended to mean a substance that may be present in a pharmaceutical composition and that is not an active ingredient, such as a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring or coloring agent. In some embodiments, suitable pharmaceutical excipients to be added to the LNP composition may include, for example, salts, starches, dextrose, lactose, sucrose, gelatin, sodium chloride, glycerol, propylene glycol, ethylene glycol, water, ethanol, and the like.

Encoding: as used herein, the term "encoding" refers to sequence information that directs the production of a first molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence of a second molecule. For example, a DNA molecule may encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase). RNA molecules can encode polypeptides (e.g., by a translation process). Thus, if transcription and translation of an mRNA corresponding to a gene produces a polypeptide in a cell or other biological system, the gene, cDNA, or single-stranded RNA (e.g., mRNA) encodes the polypeptide. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand whose nucleotide sequence is identical to the mRNA sequence of such target polypeptide agent. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such target polypeptide agent, which can be used as a template for transcription of a gene or cDNA. As understood in the art, the phrase "nucleic acid encoding a peptide or protein" means that the nucleic acid, if present in a suitable environment, e.g., in a cell and/or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a translation process. In some embodiments, the coding is capable of interacting with cellular translation mechanisms, thereby allowing translation of such coding RNAs to produce the encoded peptides or proteins.

Endogenous: as used herein, "endogenous" refers to substances from or produced within an organism, cell, tissue, or system in which it is found.

Exogenous: as used herein, the term "exogenous" refers to a substance that is produced in or outside of the organism, cell, tissue, or system in which it is located.

Expression: as understood in the art, the term "expression" is used to refer to the production of a templated nucleic acid (typically an RNA template) and/or a polypeptide encoded thereby. Thus, in some embodiments, the term may be used to refer to the production of RNA, polypeptides, RNAs, and polypeptides; alternatively or additionally, in some embodiments, it may refer to partial expression of a nucleic acid. Furthermore, expression may be transient or stable or continuous. As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by splicing, editing, 5 'cap formation, and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein. Those skilled in the art will appreciate that the term "expression" or "translation" when applied to RNA generally refers to the process by which a ribosome (e.g., in a cell) reads the strand encoding the RNA (e.g., messenger RNA) and directs the assembly of amino acid sequences to make the encoded peptide or protein.

Expression control sequence: as will be appreciated by those of skill in the art upon reading this disclosure, the term "expression control sequence" as used herein refers to a sequence element whose presence and/or identity affects one or more characteristics of the expression of another sequence. Typically, expression control sequences are nucleic acid sequence elements and are typically in cis-acting. Thus, in some embodiments, the expression control sequence may be, for example, a promoter, an enhancer, a repressor element, a loop-forming site, a termination site, a ribosome binding sequence, a translation suspension signal, and/or another control element that, for example, can control or regulate transcription of a gene and/or translation of transcribed RNA. In certain embodiments of the invention, expression control sequences may be modulated. The precise structure of the expression control sequences present in and/or otherwise associated with a particular expressible construct can vary, for example, depending on the species or cell type of the relevant expression machinery (e.g., RNA polymerase, spliceosome, ribosome, etc.), but in many embodiments can include 5' -non-transcribed and 5' -and 3' -non-translated sequences involved in initiating transcription and translation, respectively. More specifically, in some embodiments, the 5' -non-transcriptional expression control sequence may include a promoter region comprising a promoter sequence for transcriptional control of a functionally linked gene. In some embodiments, the expression control sequences may also include an enhancer sequence or an upstream activator sequence. In many embodiments, the expression control sequences of the DNA molecules may include 5' -non-transcribed sequences and 5' -and 3' -non-translated sequences, such as TATA boxes, capping sequences, CAAT sequences, and the like.

Fed-batch process: the term "fed-batch process" as used herein refers to a process in which one or more components are introduced into a vessel (e.g., a bioreactor) at some time after the reaction begins. In some embodiments, one or more components are introduced by a fed-batch process to maintain their low concentrations during the reaction. In some embodiments, one or more components are introduced by a fed-batch process to replenish the components depleted during the reaction.

5' -terminal untranslated region: as used herein, the term "5 'untranslated region" or "5' utr" refers to the sequence of an mRNA molecule that begins at the transcription initiation site and ends one nucleotide (nt) before the start codon (typically AUG) of the RNA coding region.

Fragments: "fragment" with respect to a nucleic acid sequence refers to a portion of the nucleic acid sequence, e.g., a sequence representing less than the parent sequence from which the fragment was derived, e.g., a nucleic acid sequence shortened at the 5 'and/or 3' ends and/or obtained by removal of one or more internal residues. In some embodiments, fragments of a nucleic acid sequence comprise at least 80% or in some embodiments at least 90%, 95%, 96%, 97%, 98% or 99% of the corresponding nucleotide residues from such a parent nucleic acid sequence. In many embodiments, the fragment retains one or more properties or attributes of its parent sequence. For example, in some embodiments, a fragment of a translatable RNA is characterized by stability and/or translation efficiency at least comparable to its parent. In some embodiments, a nucleic acid whose nucleic acid sequence represents two or more discrete sequences derived from the same parent nucleic acid fused together is considered a fragment of that parent nucleic acid.

"fragment" in reference to an amino acid sequence (peptide or protein) refers to a portion of an amino acid sequence, e.g., a sequence representing an amino acid sequence that is shortened and/or deleted at the N-terminal and/or C-terminal end of one or more internal residues. In some embodiments, the fragment shortened at the C-terminus (N-terminal fragment) may be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. In some embodiments, the shortened fragment at the N-terminus (C-terminal fragment) may be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, so long as the truncated open reading frame comprises the start codon for initiating translation. In many embodiments, a fragment of an amino acid sequence comprises, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. In many embodiments, the fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids. In many embodiments, the fragment retains one or more properties or attributes of its parent sequence. In some embodiments, polypeptides whose nucleic acid sequences represent two or more discrete sequences derived from the same parent polypeptide fused together are considered fragments of that parent polypeptide.

Functionality: as used herein, a "functional" biomolecule is a form of a biomolecule that exhibits its characterized properties and/or activity. In some embodiments, the biomolecule may have one function (i.e., single function), two functions (i.e., dual function), or multiple functions (i.e., multi-function).

Functional analogues: in certain embodiments, an "analog" is a "functional analog". The term "functional analogue" refers to an analogue of a substance comprising one or more functions or sharing one or more functions with a reference substance. For example, a functional analog of Nucleoside Triphosphates (NTPs) shares one or more functions with a reference NTP. For example, functional analogues of GTP share one or more functions with GTP. For example, a functional analog of CTP shares one or more functions with CTP. For example, functional analogs of ATP share one or more functions with ATP. For example, functional analogs of UTP share one or more functions with UTP. In certain embodiments, the functional analog of NTP is translatable. In certain embodiments, functional analogs of NTP may be incorporated into the product molecule (e.g., into RNA) in place of, i.e., in place of, the reference NTP. In certain embodiments, the functional analog of NTP, when incorporated into an RNA molecule, allows for translation of the RNA molecule, wherein the functional analog functions as a reference NTP during translation. In some embodiments, the functional analog of the NTP has other features not shared with the reference NTP; for example, it is known that the incorporation of pseudo-UTP and/or N1-methyl pseudo-UTP instead of, i.e., in place of, UTP may result in reduced immunogenicity of RNA as compared to RNA transcribed from the same template using unmodified UTP. In some embodiments, functional analogs of UTP may be incorporated into RNA molecules in place of UTP and/or the functional analogs are translatable, for example as UTP or in place of UTP. Similarly, functional analogues of GTP may be incorporated into RNA molecules in place of GTP and/or the functional analogues may be translatable, for example as GTP or in place of GTP. The same applies to CTP and ATP. In certain embodiments, functional analogs of the NTP may be incorporated at any given location where the corresponding NTP is expected or predicted during synthesis of the RNA molecule, for example by the substrate used, such as DNA from which the RNA is transcribed.

Functional linkage: as used herein, "functional links" or "functional connections" relate to connections within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if the promoter affects transcription of the coding sequence. Functionally linked nucleic acids are typically adjacent to each other, separated by additional nucleic acid sequences where appropriate, and in particular embodiments transcribed by an RNA polymerase to produce a single RNA molecule (co-transcript). In particular embodiments, the nucleic acid is functionally linked according to the invention to an expression control sequence, which may be homologous or heterologous with respect to the nucleic acid.

Gene: as used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and/or a polypeptide product). In some embodiments, the gene comprises a coding sequence (i.e., a sequence encoding a particular product); in some embodiments, the gene comprises a non-coding sequence. In some particular embodiments, a gene may include both coding sequences (e.g., exons) and non-coding sequences (introns). In some embodiments, a gene may include one or more regulatory elements that, for example, may control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.).

Gene product or expression product: as used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (pre-and/or post-processing) or a polypeptide encoded by RNA transcribed from a gene (pre-and/or post-modification).

Heterologous: the term "heterologous" is used herein to describe an entity relative to a reference and designates the entity as originating from a source that is different from the relevant reference and/or associated with one or more components other than the relevant reference. As one example, introducing cells of one individual into a different individual constitutes a xenograft. The heterologous gene is a gene derived from a source other than the subject.

Homology: the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at the corresponding positions). For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids that are similar to one another, and/or have "polar" or "nonpolar" side chains, as is well known to those of ordinary skill in the art. Substitution of one amino acid for another of the same type may be generally considered a "homologous" substitution.

Host cell: as used herein, refers to a cell into which an exogenous substance (e.g., DNA, such as recombinant or other means) has been introduced. The skilled artisan will appreciate upon reading this disclosure that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the host cell includes prokaryotic and eukaryotic cells selected from any life kingdom suitable for expression of exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic cells (single or multiple cells), bacterial cells (e.g., E.coli), bacillus (Bacillus spp.), streptomyces (Streptomyces spp), etc.), mycobacterial cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae, schizosaccharomyces pombe (S.pombe), pichia pastoris, pichia methanolica (P.methyotis), etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus infected insect cells, trichoplusia ni (Trichoplusia ni), etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or tetrahybridomas). In some embodiments, the host cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the host cell is a eukaryotic cell. For example, the eukaryotic host cell may be CHO (e.g., CHO Kl, DXB-1 CHO, veggie-CHO), COS (e.g., COS-7), retinal cells, vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, haK, BHK), heLa, hepG2, WI38, MRC 5, colo205, HB 8065, HL-60 (e.g., BHK 21), jurkat, daudi, A (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived therefrom.

Hybridization: two sequences are "capable of hybridizing" or "hybridize" if one nucleic acid and the other nucleic acid are complementary to each other. Two sequences are "complementary" if one nucleic acid and the other nucleic acid are capable of forming a stable duplex with each other, e.g., hybridize to each other to form a double-stranded molecule. Complementarity may be complete or partial. Those skilled in the art will appreciate that the ability of two sequences to hybridize to each other may depend on conditions (e.g., temperature, pH) and/or the presence of other potentially competing sequences. In some embodiments, hybridization is performed under stringent conditions, so that only highly complementary sequences form stable hybrids. Exemplary such stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J.Sambrook et al, editions, 2 nd edition, cold Spring Harbor Laboratory press, cold Spring Harbor, new York,1989 or Current Protocols in Molecular Biology, F.M. Ausubel et al, editions, john Wiley & Sons, inc., new York. For example, in some embodiments, stringent hybridization can involve incubating the hybridized nucleic acid with a membrane containing complementary nucleic acid in hybridization buffer (3.5 XSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA) at 65 ℃. SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7; after such incubation, the membrane of the transferred DNA is washed, for example, in 2 XSSC at room temperature, and then in 0.1-0.5 XSSC/0.1 XSDS at a temperature of up to 68 ℃.

Identity: as used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymer molecules are considered "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. The calculation of the percent identity of two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package using the nwsgapdna.

Improvement, increase or decrease: as used herein, these terms, or grammatically comparable comparison terms, refer to values measured relative to a comparable reference. For example, in some embodiments, the evaluation value obtained with an agent of interest may be "improved" relative to the evaluation value obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, the evaluation value obtained in the subject or system of interest may be "improved" relative to an evaluation value obtained in the same subject or system under different conditions (e.g., before or after an event such as administration of an agent of interest) or in a different comparable subject (e.g., in the presence of one or more indicators of a particular disease, disorder, or condition of interest in a comparable subject or system different from the subject or system of interest, or prior exposure to a disorder or agent, etc.). In some embodiments, the term comparison refers to a statistically relevant difference (e.g., a prevalence and/or magnitude sufficient to achieve a statistical correlation). In a given context, those skilled in the art will recognize or will be able to readily determine the degree and/or prevalence of the differences required or sufficient to achieve such statistical significance.

Increase and enhancement: as understood in the art, terms such as "increasing" or "enhancing" may be used herein to refer to, for example, an overall and/or relative increase or enhancement of an entity, event, frequency, activity, or the like. In some embodiments, the increase may be, for example, about at least 10%, in some embodiments at least 20%, in some embodiments at least 30%, in some embodiments at least 40%, in some embodiments at least 50%, 55%, 65%, 70%, 75%, in some embodiments at least 80%, 85%, 90%, 95%, and in some embodiments at least 100% or more. In some embodiments, the increase or enhancement may be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

In vitro: the term "in vitro" as used herein refers to events that occur in an artificial environment (e.g., in a test tube or reaction vessel (e.g., bioreactor), in a cell culture, etc.) rather than within a multicellular organism.

In vitro transcription, transcription: as known in the art, the term "transcription" refers to the process by which a nucleic acid molecule having a specific nucleic acid sequence ("nucleic acid template") is read by an RNA polymerase such that the RNA polymerase synthesizes its complementary single stranded RNA molecule. During transcription, genetic information in the nucleic acid template is transcribed. In some embodiments, the nucleic acid template is or comprises DNA; however, in some embodiments, for example in the case of transcription from an alphavirus nucleic acid template, the nucleic acid template may be or comprise RNA. In some embodiments, a nucleic acid template may include one or more residues that are neither DNA nor RNA and/or are DNA or RNA analogs (e.g., contain one or more modifications, such as backbone modifications or base modifications) relative to canonical DNA or RNA. In many embodiments, transcribed RNA can be translated into protein. In many embodiments, the term "transcription" as used herein refers to "in vitro transcription" as will be clear from the context. As used herein, the term "in vitro transcription" or "IVT" refers to a process in which transcription occurs in vitro (i.e., outside an organism and typically in a non-cellular system) to produce a synthetic RNA product; in many embodiments, the disclosure describes IVTs that generate RNA products for use in certain applications, including, for example, the production of proteins or polypeptides. In some embodiments, the RNA product produced may be translated in vitro, or may be introduced directly into a cell, where in some embodiments it may be translated. In certain specific embodiments, the RNA product produced is of sufficient size and/or quality to be administered to an organism, and in some embodiments, to a human (e.g., as a pharmaceutically active RNA). In some embodiments, the RNA product can be selected from, for example, but not limited to, mRNA, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNAs (e.g., for CRISPR), ribosomal RNAs, micronuclear RNAs, micronucleolar RNAs, and the like. The IVT reaction typically utilizes a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotides or modified ribonucleotides triphosphates), and an appropriate RNA polymerase as described and/or utilized herein. In some embodiments, cloning vectors are used to generate transcripts. In some such embodiments, the cloning vector is designated as a "transcription vector" (which is encompassed by the term "vector" according to the present invention). In some embodiments, the cloning vector may be a plasmid. In some embodiments, the RNA is in vitro transcribed RNA (IVT-RNA) and is obtainable by in vitro transcription of an appropriate DNA template. Those skilled in the art will appreciate a variety of promoter sequences that may be suitably used to control transcription of the relevant RNA polymerase. In some embodiments, a DNA template for in vitro transcription may be obtained by cloning a nucleic acid, such as a cDNA, and introducing it into an appropriate vector for in vitro transcription. In some embodiments, the cDNA may be obtained by reverse transcription of RNA.

In vitro transcribed RNA composition: as used herein, the term "in vitro transcribed RNA composition" refers to a composition comprising RNA synthesized by in vitro transcription. In some embodiments, such compositions may comprise an excess of in vitro transcription reagents (including, for example, ribonucleotides and/or capping agents), nucleic acids or fragments thereof (such as DNA templates or fragments thereof), polypeptides or fragments thereof (such as recombinases or host cell proteins or fragments thereof), and/or other impurities. In some embodiments, the in vitro transcribed RNA composition may have been treated and/or processed prior to the purification process that ultimately produces an RNA transcript formulation comprising the RNA transcript at the desired concentration in an appropriate buffer for formulation and/or further manufacture and/or processing. For example, in some embodiments, the in vitro transcribed RNA composition may have been treated to remove or digest DNA templates (e.g., using DNase). In some embodiments, the in vitro transcribed RNA composition may have been treated to remove or digest polypeptides (e.g., enzymes such as RNA polymerase, RNase inhibitors, etc.) present in the in vitro transcription reaction (e.g., using proteases). Thus, in some embodiments, after transcription of the RNA, the DNA template may be removed or isolated from the composition comprising the RNA; those skilled in the art are aware of the variety of methods by which such removal can be accomplished, such as DNA hydrolysis. In some embodiments, an RNase inhibitor may be added during DNA removal or digestion to protect RNA from potential degradation. In some embodiments, the in vitro transcribed RNA composition may have been treated to remove or digest peptides (e.g., enzymes such as RNA polymerase, RNase inhibitors, etc.) present in the in vitro transcription reaction (e.g., using proteases).

In vivo: as used herein, the term "in vivo" refers to events that occur within multicellular organisms (such as humans and non-human animals).

Separating: the term "isolated" as used herein generally refers to a molecule or other entity that is substantially free of other components (such as other cellular material); in some embodiments, an "isolated" entity is substantially free of components with which it was previously associated (e.g., when initially produced). In certain embodiments, the term "isolated nucleic acid" as used herein refers to a nucleic acid that has been subjected to the following treatments: (i) in vitro amplification, for example by Polymerase Chain Reaction (PCR), (ii) production by clonal recombination, (iii) fractionation purification, for example by cleavage and gel electrophoresis, or (iv) synthesis, for example by chemical synthesis or IVT synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid that can be used for manipulation by recombinant techniques.

And (3) connection and fusion: as used herein, the terms "linked" or "fused" are used interchangeably. These terms refer to two or more elements or components or domains (e.g., domains from two different proteins or nucleic acid molecules) that are bound together (e.g., by covalent linkages).

Messenger RNA, mRNA: according to the present invention, the term "mRNA" means "messenger RNA" and refers to transcripts that are typically generated from a template (e.g., a DNA template) and encode a peptide or protein. Typically, the mRNA comprises 5'UTR, protein coding region, 3' UTR and poly (A) sequences. In some embodiments, mRNA can be generated from a DNA template as described herein by in vitro transcription. In some embodiments, the mRNA may be modified, for example, by stable modification and/or capping. In some embodiments, a nucleic acid (such as RNA, e.g., mRNA) may encode a peptide or protein. Thus, in some embodiments, a transcribable nucleic acid sequence or transcript thereof may contain an Open Reading Frame (ORF) encoding a peptide or protein.

Nanoparticles: as used herein, the term "nanoparticle" refers to particles having a diameter of less than 1000 nanometers (nm). In some embodiments, the nanoparticle has a diameter of less than 300nm, as defined by the national science foundation. In some embodiments, the nanoparticle has a diameter of less than 100nm, as defined by the national institutes of health. In some embodiments, the nanoparticle has a diameter of less than 80nm, as defined by the national institutes of health. In some embodiments, the nanoparticle comprises one or more closed compartments separated from the bulk solution by a membrane that encloses and closes the space or compartment.

Nucleic acid/polynucleotide: the term "nucleic acid" as used herein refers to a polymer comprising two or more nucleotide or nucleotide analogue residues. In some embodiments, a nucleic acid may include one or more residues or linkages that are modified relative to naturally occurring DNA or RNA residues. For example, in some embodiments, a nucleic acid may have one or more modifications of bases, sugars, or backbones (e.g., phosphate) relative to naturally occurring DNA or RNA residues. In some embodiments, a nucleic acid molecule refers to or comprises a nucleic acid that is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the nucleic acid may be or comprise or may have sequences found in molecules of genome DNA, cDNA, mRNA, virus RNA, siRNA, miRNA, shRNA, recombinant preparation and chemical synthesis. In some embodiments, the term "nucleic acid" refers to a polymer of at least 2 residues or more (including, for example, at least 3 residues, at least 4 residues, at least 5 residues, at least 6 residues, at least 7 residues, at least 8 residues, at least 9 residues, at least 10 residues, or more). In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a Peptide Nucleic Acid (PNA). In some embodiments, the nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double stranded nucleic acid. In some embodiments, the nucleic acid comprises single-stranded and double-stranded portions. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5' -N-phosphoramidite linkages and/or one or more peptide linkages, e.g., as in "peptide nucleic acids". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all non-natural residues. In some embodiments, the unnatural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, 1-methyl-pseudouridine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on complementary templates, e.g., in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1, 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,19,000, 19,000, or more nucleotides in length.

Nucleic acid sequence: according to the present invention, "nucleic acid sequence" refers to a sequence of residues in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the term is used to refer to the sequence of the entire nucleic acid molecule (such as a single strand of the entire nucleic acid molecule); in some embodiments, the term is used to refer to a sequence that represents a portion (e.g., fragment) thereof.

Nucleotide: the term "nucleotide" is used herein as generally understood in the art and may refer to nucleoside monophosphates, nucleoside diphosphate and nucleoside triphosphates.

Pharmaceutical grade: the term "pharmaceutical grade" as used herein refers to standards of chemical and biological drug substances, pharmaceutical products, dosage forms, composite formulations, excipients, medical devices, and dietary supplements established by the accepted national or regional pharmacopoeia (e.g., the united states pharmacopeia and the formulary (USP-NF)).

Polypeptide: the term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids or more. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be generic enough to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains (e.g., fragments, portions, or domains that retain at least one activity) of such complete polypeptides. In some embodiments, the polypeptide may contain an L-amino acid, a D-amino acid, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise a peptidomimetic). In some embodiments, the polypeptide may be or comprise an enzyme. In some embodiments, the polypeptide may be or comprise a polypeptide antigen. In some embodiments, the polypeptide may be or comprise an antibody agent. In some embodiments, the polypeptide may be or comprise a cytokine.

Primary structure: the term "primary structure" as used herein with respect to nucleic acid molecules refers to the linear sequence of monomer residues.

Promoter, promoter region: the term "promoter" or "promoter region" refers to a nucleic acid sequence that directs the synthesis of a transcript (e.g., a transcript comprising a coding sequence), for example, by providing recognition and binding sites for an RNA polymerase. In some embodiments, the promoter region may include other recognition or binding sites for other factors involved in regulating transcription of the gene. In some embodiments, the promoter may control transcription of a prokaryotic or eukaryotic gene. In some embodiments, the promoter may be "inducible" and initiate transcription in response to an inducer; in some embodiments, a promoter may be "constitutive" if transcription is not under the control of an inducer or a cell type specific promoter. In some embodiments, the inducible promoter is expressed only to a very small extent or not at all if the inducer is not present; when an inducer is present, the promoter is "turned on" or transcription level is increased, typically mediated by binding of a particular transcription factor.

Pure or purified: as used herein, an agent or entity is "pure" or "purified" if it is substantially free of other components. For example, a formulation containing more than about 90% of a particular agent or entity is generally considered to be a pure formulation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure in the formulation.

Ribonucleotides: as used herein, the term "ribonucleotide" includes unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides (analogs) can include one or more modifications including, but not limited to, for example, (a) terminal modifications such as 5 'terminal modifications (e.g., phosphorylations, dephosphorations, conjugates, reverse linkages, etc.), 3' terminal modifications (e.g., conjugates, reverse linkages, etc.), (b) base modifications such as base substitutions with modified bases, stabilized bases, destabilized bases, or bases that base pair with all components of the amplified partner or conjugated bases, (c) sugar modifications (e.g., at the 2 'position or the 4' position) or sugar substitutions, and (d) internucleoside linkages modifications including modifications or substitutions of phosphodiester linkages. In some embodiments, the modified ribonucleotide retains at least one function of the corresponding unmodified ribonucleotide. The term "ribonucleotide" may include ribonucleotides that are triphosphate, including modified and unmodified ribonucleotides.

Ribonucleic acid (RNA): as used herein, the term "RNA" refers to a polymer of ribonucleotides; the term "RNA" or "RNA molecule" relates to a molecule comprising ribonucleotide residues. In some embodiments, an "RNA" is composed entirely or substantially of ribonucleotide residues. As known to those skilled in the art, the specification "ribonucleotide" is a nucleotide that has a hydroxy group at the 2' -position of the β -D-ribofuranosyl group. The term "RNA" may include double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or fully purified RNA), substantially pure RNA, synthetic RNA, and recombinantly produced RNA (such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides). In some embodiments, the RNA can be modified relative to a reference (e.g., naturally occurring RNA) for example by adding non-nucleotide species, such as to the end or interior of the RNA, e.g., at one or more nucleotides of the RNA. In some embodiments, one or more residues or linkages in the RNA molecule may be or comprise non-standard residues or linkages, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides; in some embodiments, such RNAs may be referred to as analogs, e.g., analogs of naturally occurring RNAs. In some embodiments, the RNA is single stranded. In some embodiments, the RNA is double stranded. In some embodiments, the RNA comprises both single-stranded and double-stranded portions. In some embodiments, the RNA can comprise a backbone structure as described above in the definition of "nucleic acid/polynucleotide". The RNA can be a regulatory RNA (e.g., siRNA, microrna, etc.) or a messenger RNA (mRNA). In some embodiments, wherein the RNA is mRNA. In some embodiments where the RNA is mRNA, the RNA typically comprises a poly (a) region at its 3' end. In some embodiments where the RNA is mRNA, the RNA typically comprises a cap structure at its 5' end that is recognized in the art, for example, to recognize the mRNA and attach it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNAs include RNAs synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods). In some embodiments, the RNA is single stranded RNA. In some embodiments, the single stranded RNA may comprise self-complementing elements and/or may establish secondary and/or tertiary structures. The term "single stranded RNA" will be understood by those skilled in the art to generally refer to an RNA molecule that has no complementary nucleic acid molecules (typically no complementary RNA molecules) associated therewith. In some embodiments, the single stranded RNA may contain self-complementary sequences that allow portions of the RNA to fold back and form secondary structural motifs, including but not limited to base pairs, stems, stem loops, and/or projections as known in the art. Those of skill in the art will understand from the context whether a single stranded RNA refers to a coding strand sequence or its complement when referred to as "coding. In some embodiments, the single-stranded RNA may be self-amplified RNA (also referred to as self-replicating RNA).

Recombination: as used herein, the term "recombinant" when used in reference to a polypeptide is intended to refer to a polypeptide designed, engineered, prepared, expressed, produced, manufactured, and/or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise manipulated to express one or more genes or gene components encoding and/or directing the expression of the polypeptide or one or more components, portions, elements, or domains thereof; and/or by any other means related to splicing or ligating selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements, and/or otherwise generating nucleic acids encoding and/or directing expression of the polypeptide or one or more components, portions, elements or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are computer-designed. In some embodiments, one or more such selected sequence elements are produced by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources in a germline such as a source organism of interest (e.g., human, mouse, etc.). In some embodiments, the nucleic acids described herein may be recombinant and/or isolated molecules.

Reference is made to: as used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and/or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, a reference or control is determined or characterized under conditions or conditions comparable to the evaluation conditions or conditions, as will be understood by those skilled in the art. Those skilled in the art will understand when sufficient similarity exists to justify reliance on and/or comparison with a particular possible reference or control.

RNA polymerase: as used herein, the term "RNA polymerase" refers to an enzyme that catalyzes the synthesis of polynucleic nucleotides by adding ribonucleotide units to a nucleotide chain using DNA or RNA as a template. As will be clear from the context, the term refers to the complete enzyme that is present in nature, or an isolated active catalytic or functional domain or fragment thereof. In some embodiments, the RNA polymerase initiates synthesis at the 3 'end of the primer or nucleic acid strand or at the promoter sequence, and proceeds in the 5' direction along the target nucleic acid to synthesize a strand complementary to the target nucleic acid until synthesis is terminated.

RNA transcript preparation: the term "RNA transcript formulation" as used herein refers to a formulation comprising RNA transcripts purified from the in vitro transcribed RNA compositions described herein. In some embodiments, the RNA transcript formulation is a formulation comprising a pharmaceutical grade RNA transcript. In some embodiments, the RNA transcript formulation is a formulation comprising an RNA transcript that has one or more product quality attributes characterized and determined to meet release and/or acceptance criteria (e.g., as described herein). Examples of such product quality attributes include, but are not limited to, appearance, RNA length, characteristics of the drug substance as RNA, RNA integrity, RNA sequence, RNA concentration, pH, osmolality, residual DNA template, residual double stranded RNA, bacterial endotoxin, bioburden, and combinations thereof.

Room temperature: as used herein, the term "room temperature" refers to the ambient temperature. In some embodiments, room temperature is about 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, preferably about 18 ℃ to 30 ℃, such as about 18 ℃ to 25 ℃, or about 20 ℃ to 30 ℃, or about 23 ℃ to 27 ℃, or about 25 ℃.

Sample: as used herein, the term "sample" generally refers to an aliquot of a material obtained or derived from a source of interest, e.g., as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a mouse). In some embodiments, the source of interest is or includes biological tissue or fluid. In some embodiments, the biological fluid may be or include an intracellular fluid, an extracellular fluid, an intravascular fluid (plasma), a interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, the biological tissue or sample may be obtained by, for example, aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing, or lavage (e.g., bronchoalveolar, catheter, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, the sample is or includes cells obtained from the subject. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a formulation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and/or by adding one or more agents thereto). For example, a "treated sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acids, isolation and/or purification of certain components, and the like.

Secondary structure: as understood in the art, the term "secondary structure" is used to refer to interactions between bases in a nucleic acid molecule. Thus, the secondary structure can be described as a two-dimensional representation of a nucleic acid molecule reflecting base pairing. The term is generally used to refer to intramolecular base pairing interactions in a single stranded molecule (e.g., single stranded RNA). In fact, many single stranded nucleic acid molecules, particularly single stranded RNA molecules, are characterized by (intramolecular) base pair regions. According to the present invention, the term "secondary structure" encompasses structural motifs including, but not limited to, base pairs, stems, stem loops, projections, loops (such as inner loops and multi-branched loops). The secondary structure of a nucleic acid molecule can be represented by a two-dimensional map (plan view) showing base pairing (see Auber et al, (2006), J.Graph Algorithms appl.,10:329-351 for more details on the secondary structure of an RNA molecule). As described herein, the secondary structure of certain RNA molecules is relevant in the context of the present invention. The secondary structure of a nucleic acid molecule, in particular a single-stranded RNA molecule, can be determined by prediction using a web server (http:// RNA. Urmc. Rochester. Edu/RNAstructureWeb/Servers/prediction 1. Html) for RNA secondary structure prediction.

Stable: the term "stable" when applied to nucleic acids and/or compositions comprising nucleic acids (e.g., encapsulated in lipid nanoparticles) means that such nucleic acids and/or compositions retain one or more aspects of their characteristics (e.g., physical and/or structural features, functions, and/or activities) over a period of time under a specified set of conditions (e.g., pH, temperature, light, relative humidity, etc.). In some embodiments, this stability is maintained for a period of at least about one hour; in some embodiments, this stability is maintained for a period of time of about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty four (24) months, about thirty six (36) months, or more. In some embodiments, this stability is maintained over a period of time ranging from about one (1) day to about twenty-four (24) months, from about two (2) weeks to about twelve (12) months, from about two (2) months to about five (5) months, and the like. In some embodiments, this stability is maintained at ambient conditions (e.g., at room temperature and ambient pressure). In some embodiments, this stability is maintained under physiological conditions (e.g., in vivo or at about 37 ℃, e.g., in serum or in phosphate buffered saline). In some embodiments, this stability is maintained under refrigeration (e.g., at or below about 4 ℃ C., including, for example, -20 ℃ C., or-70 ℃ C.). In some embodiments, this stability is maintained when the nucleic acid and/or the composition comprising the nucleic acid is protected from light (e.g., maintained in the dark).

As an example, in some embodiments, the term "stable" is used to refer to a nanoparticle composition (e.g., a lipid nanoparticle composition). In such embodiments, the stabilized nanoparticle composition (e.g., stabilized nanoparticle composition) and/or components thereof retains one or more aspects of its characteristics (e.g., physical and/or structural features, functions, and/or activities) over a period of time under a specified set of conditions. For example, in some embodiments, a stabilized nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized by an average particle size, particle size distribution, and/or polydispersity of the nanoparticles that is substantially maintained (e.g., within 10% or less of the initial characteristics) over a period of time (e.g., as described herein) under a specified set of conditions (e.g., as described herein). In some embodiments, the stabilized nanoparticle composition (e.g., lipid nanoparticle composition) is characterized by the absence of a detectable amount of degradation products (e.g., associated with hydrolysis and/or enzymatic digestion) after it has been maintained under a specified set of conditions (e.g., as described herein) for a period of time.

Stability of RNA: the term "stability of RNA" is generally used herein to refer to the "half-life" of RNA. "half-life" refers to the period of time required to eliminate half of the activity, amount or number of molecules. In many embodiments, the half-life of the RNA is indicative of its stability. Those skilled in the art will appreciate that the half-life of RNA can often affect the "duration of expression" of RNA; in general, an RNA with a long half-life will be expressed for an extended period of time relative to an RNA with a shorter half-life.

Stem loop, hairpin: as used herein, the term "stem loop" or "hairpin loop" refers to a particular secondary structure of a nucleic acid molecule (typically a single-stranded nucleic acid molecule, such as single-stranded RNA). The specific secondary structure represented by the stem loop consists of a contiguous nucleic acid sequence comprising a stem and a loop (e.g., a terminal loop) (also known as a hairpin loop), wherein the stem is formed by two adjacent full or partial complementary sequence elements; the sequence elements are separated by a short sequence (e.g., 3-10 nucleotides) of the loop forming the stem-loop structure. Two adjacent full or partial complementary sequences can be defined as, for example, stem loop elements stem 1 and stem 2. When these two adjacent full or partial reverse complement sequences (e.g., stem loop element stem 1 and stem 2) form a base pair with each other, a stem loop is formed, resulting in a double stranded nucleic acid sequence comprising at its ends a unpaired loop formed by a short sequence located between stem loop element stem 1 and stem 2. Thus, a stem loop comprises two stems (stem 1 and stem 2) which form base pairs with each other at the level of the secondary structure of the nucleic acid molecule and are separated by a short sequence which is not part of stem 1 or stem 2 at the level of the primary structure of the nucleic acid molecule. To illustrate, the two-dimensional representation of the stem loop resembles a lollipop-shaped structure. The formation of a stem-loop structure involves a sequence that can be folded back on itself to form a paired double strand; the paired duplex is formed by stem 1 and stem 2. The stability of the paired stem-loop element is generally determined by the length, the number of nucleotides of stem 1 that are capable of forming base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides of stem 2 relative to the number of nucleotides of stem 1 that are incapable of forming such base pairs (mismatches or bulges) with nucleotides of stem 2. If a given nucleic acid sequence is characterized by a stem loop, the corresponding complementary nucleic acid sequence is generally also characterized by a stem loop. The stem loop is typically formed from a single stranded RNA molecule.

And (3) synthesis: as used herein, the term "synthetic" refers to entities that are manufactured manually or with human intervention or that are produced synthetically rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a chemically synthesized nucleic acid molecule, e.g., by solid phase synthesis in some embodiments. In some embodiments, the term "synthetic" refers to an entity made outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) produced by in vitro transcription using a template.

And (3) a template: as used herein, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed. In some embodiments, the template is DNA. In some embodiments, the DNA template is a linear DNA molecule. In some embodiments, the DNA template is a circular DNA molecule. DNA may be obtained or generated using methods known in the art, including, for example, gene synthesis, recombinant DNA techniques, or combinations thereof. In some embodiments, the DNA template comprises a nucleotide sequence encoding a transcribed region of interest (e.g., encoding an RNA described herein), and a promoter sequence that is recognized by an RNA polymerase selected for in vitro transcription, such as the RNA polymerase described herein. In some embodiments, the DNA template encodes one or more elements of a product (such as RNA), e.g., a 5'utr, a 3' utr, an open reading frame (e.g., encoding a peptide or protein of interest, such as an antigen), a poly (a) tail, etc. In some embodiments, the DNA template encodes all elements of the product RNA. In some embodiments, the DNA template does not encode all elements of the product RNA, e.g., the DNA template may not encode a poly (a) tail, and such a poly (a) tail may be enzymatically added to the RNA after transcription as described herein.

Tertiary structure: as used herein, the term "tertiary structure" with respect to a nucleic acid molecule refers to the three-dimensional structure of the nucleic acid molecule, as defined by atomic coordinates.

3' -terminal untranslated region: as used herein, the term "3 'untranslated region" or "3' utr" refers to the sequence of an mRNA molecule that begins after the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3' utr begins immediately after the stop codon of the coding region of the open reading frame sequence. In other embodiments, the 3' utr does not begin immediately after the stop codon of the coding region of the open reading frame sequence.

Threshold level (e.g., acceptance criteria): as used herein, the term "threshold level" refers to a level that is used as a reference to obtain information about and/or classify a measurement (e.g., a measurement obtained in an assay). For example, in some embodiments, the threshold level means a value measured in an assay that defines a demarcation line between two subsets of a population (e.g., a lot that meets quality control criteria and a lot that does not meet quality control criteria). Thus, values at or above the threshold level define one subset of the population, and values below the threshold level define another subset of the population. The threshold level may be determined based on one or more control samples or across a population of control samples. The threshold level may be determined before, simultaneously with, or after the measurement of interest is made. In some implementations, the threshold level may be a range of values.

Transcription efficiency: the term "transcription efficiency" relates to the amount of transcription product produced from a template molecule over a specific period of time.

Translation efficiency: the term "translation efficiency" relates to the amount of translation product provided by an RNA molecule over a specified period of time.

Variants: the term "variant" when used in reference to, for example, nucleic acid and amino acid sequences includes any variant, particularly mutants, strain variants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those that occur naturally. Allelic variants involve alterations in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing can typically identify a large number of allelic variants of a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid according to the invention is a nucleic acid that differs in codon sequence from a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence that has a different species origin than a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence of different viral origin than the given nucleic acid or amino acid sequence. In some embodiments, a nucleic acid variant may include single or multiple nucleotide deletions, additions, mutations, substitutions, and/or insertions as compared to a reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. The addition variants comprise 5 '-and/or 3' -terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides. In the case of substitution, at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted at its position (such as a transversion and a transition). Mutations may include abasic sites, crosslinking sites, and chemically altered or modified bases. Insertion includes adding at least one nucleotide to a reference nucleic acid.

And (3) a carrier: as used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include plasmids, cosmid vectors, phagemids (such as lambda phage), viral genomes (including retroviral, adenoviral or baculovirus vectors), artificial chromosome vectors (such as Bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC) or P1 Artificial Chromosome (PAC)), and functional parts thereof. One type of vector is a "plasmid," which refers to circular double stranded DNA into which additional DNA fragments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Expression vectors include plasmids as well as viral vectors, and typically contain the desired coding sequences and appropriate non-coding sequences required for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect or mammal) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify a certain desired DNA fragment and may lack the functional sequences required to express the desired DNA fragment.

Detailed description of the preferred embodiments

Although certain embodiments of the invention are described in detail below, it is to be understood that the invention is not limited to the specific methods, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In some embodiments, practice of the invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the art literature (see, e.g., molecular Cloning: A Laboratory Manual, 2 nd edition, J.Sambrook et al, eds., cold Spring Harbor Laboratory Press, cold Spring Harbor 1989). Indeed, in many embodiments, standard techniques such as recombinant DNA production and/or manipulation, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), and the like, may be used. Those skilled in the art having read this disclosure will appreciate that enzymatic reactions and/or purification techniques may be performed according to manufacturer's instructions or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and/or as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., green and Sambrook, molecular Cloning: A Laboratory Manual (4 th edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2012)), which is incorporated herein by reference for any purpose.

In the following description, certain elements of the present application will be described. These elements may be discussed in connection with a particular embodiment, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and specific embodiments should not be construed as limiting the application to only the explicitly described embodiments. The description should be understood to disclose and cover embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred elements. Moreover, any arrangement or combination of all the described elements in this application should be considered as disclosed by the specification unless the context indicates otherwise.

Several documents are cited throughout the present specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, descriptions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the application is not entitled to antedate such disclosure by virtue of prior application.

Batch, fed-batch

The present disclosure provides certain techniques for producing RNA molecules, particularly via In Vitro Transcription (IVT) using a "batch" reaction. Those skilled in the art will appreciate that the term "batch" or "batch reaction" or similar terms are used to refer to a reaction, such as a transcription reaction, in which at least one discrete replenishment event occurs for at least one component (e.g., for at least one NTP such as UTP and/or GTP or a functional analogue thereof, and optionally for one or more other components (e.g., components of the reaction mixture discussed herein), optionally multiple components replenished in the same discrete replenishment event. In some embodiments, replenishing the reaction mixture comprises replenishing UTP or a functional analog thereof. In some embodiments, replenishing the reaction mixture comprises replenishing UTP or a functional analog thereof and GTP or a functional analog thereof. In some embodiments, replenishing the reaction mixture comprises replenishing additional components (such as ATP or a functional analog thereof and/or CTP or a functional analog thereof and/or one or more salts and/or one or more enzymes such as a polymerase and/or one or more 5' cap nucleotides and/or one or more other components of the reaction mixture described herein such as transcription buffers, RNase inhibitors, DNA templates, 5' caps and/or 5' cap analogs, etc.). In some embodiments, the reaction mixture is replenished more than once during the transcription and/or capping reaction.

In certain embodiments, a fed-batch process is used in a method of producing RNA or a composition comprising RNA. The term "fed-batch process" or "fed-batch reaction" or similar terms refer to a process or reaction in which a portion or all of the components are present in the starting reaction mixture (batch reaction) and in which the reaction is occasionally supplemented with one or more components during the course of the reaction. In some embodiments, one or more components such as UTP or a functional analog thereof and/or GTP or a functional analog thereof (e.g., introduced by a fed-batch process) are supplemented to maintain their low concentrations or to restore the ratio of their initial concentrations to the initial concentrations of CTP and/or ATP or a functional analog thereof during the reaction. In some embodiments, one or more components such as UTP or a functional analog thereof and/or GTP or a functional analog thereof (e.g., introduced by a fed-batch process) are supplemented to supplement the components depleted during the reaction. "replenishing the reaction mixture" means replenishing the components into the reaction in discrete amounts after the reaction has begun. However, the fed-batch reaction is not limited to being fed in discrete amounts. In some embodiments, replenishing includes replenishing by continuous flow, i.e., continuously replenishing one or more components of the reaction mixture during transcription and/or capping reactions.

In some embodiments, the fed-batch process involves the use of an initial reaction mixture in which all nucleotide triphosphates are present as part of the RNA to be synthesized; in other embodiments, the fed-batch process comprises using an initial reaction mixture in which not all of the nucleotide triphosphates that are part of the RNA to be synthesized are present.

In some embodiments, the starting reaction mixture contains ATP, GTP, CTP and UTP or a functional analog thereof. In some embodiments, the starting reaction mixture used is substantially free of ATP or a functional analog thereof. In some embodiments, the starting reaction mixture is substantially free of GTP or a functional analog thereof. In some embodiments, the starting reaction mixture is substantially free of CTP or a functional analog thereof. In some embodiments, the starting reaction mixture is substantially free of UTP or a functional analog thereof. It will be appreciated that when it is predicted (e.g., depending on the template used) that the RNA to be synthesized contains a component that is not present in the starting reaction mixture, that component must be replenished to synthesize the RNA.

In capping and transcription reactions, for example, the reaction begins when an RNA polymerase mediates the formation of a covalent bond between a nucleotide and a cap analogue. It will be appreciated that one distinction between capping and transcription reactions and uncapped transcription reactions is the presence of a component that provides a cap structure to the 5' end of the transcript, such as the 5' cap or 5' cap analogue described herein.

The present disclosure provides techniques involving fed-batch processes in which at least one component of the reaction mixture is present in a limited amount. By "limiting amount" is meant limiting the reaction components to limit the time until the reaction is stopped, the amount of one or more of the amount of product and transcription rate produced until the reaction is stopped (e.g., the starting concentration). For example, in some embodiments, limiting the amount of a reaction component (e.g., UTP or a functional analog thereof and/or GTP or a functional analog thereof) limits one or more of the time until transcription and/or capping reactions cease, the amount of product (e.g., RNA) produced until transcription and/or capping reactions cease, and/or the rate of transcription and/or capping reactions (e.g., the rate at which reactants are converted to product).

In some embodiments, one or more reaction components are added continuously to the reaction, such as by a fed-batch process, and one or more components, such as one or more limiting components, are added periodically to the reaction. In certain embodiments, components such as restriction components (e.g., restriction nucleotides) are periodically or intermittently replenished into the reaction by a fed-batch process. The term "periodic" means "interval occurring", which may be "regular", meaning that the characteristic is "fixed" with respect to, for example, time and/or concentration levels in the reaction. The term "intermittent" means "intermittent". "spacing" refers to both regular and irregular spacing. It should be understood that "intermittent" replenishment of the components may also be "periodic". It is also understood that intermittent introduction or replenishment of a component into a reaction means at least one time, while "periodic" introduction or replenishment of a component is at least two times (to define "regular intervals"). In some embodiments, a component (such as a restriction component, e.g., UTP and/or GTP or a functional analog thereof) may be supplemented, at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 times or more, or any range derivable therein, during transcription and/or capping reactions. In some embodiments, UTP or a functional analog thereof is supplemented at least once. In some embodiments, UTP or a functional analog thereof is supplemented at least twice. In further embodiments, UTP or a functional analog thereof is intermittently or periodically introduced into the reaction between three and 50 times. In some embodiments, such periodic replenishment may be performed as one or more boluses or batch additions (including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more boluses or batch additions). In some embodiments, such periodic replenishment may be performed by a fed-batch process. Supplementation may also include supplementation of a composition comprising UTP or a functional analogue thereof and comprising additional components such as buffers, polymerases, CTPs or functional analogues thereof, GTP or functional analogues thereof, ATP or functional analogues thereof, or other components that may be present in a transcription reaction mixture as described herein. In some embodiments, the composition for supplementation is substantially free of CTP and/or ATP or a functional analog thereof.

DS RNA contaminants

As is known in the art, during synthesis of mRNA by a transcription reaction (e.g., in Vitro Transcription (IVT), e.g., using T7 RNA polymerase or another RNAP), significant amounts of abnormal products, including double-stranded RNA (dsRNA), can be produced due to the unusual activity of enzymes (e.g., involving hybridization of different RNA molecules). dsRNA has been shown to induce inflammatory cytokines and activate effector enzymes, thereby inhibiting protein synthesis.

As described herein, dsRNA contamination of in vitro transcription reactions can be problematic. At least two different types of dsRNA contaminants have been described: (i) Short dsRNA, wherein the antisense fragment base pairs with an RNA transcript of interest (e.g., an mRNA product), and (ii) nearly full-length dsRNA. Both may be generated by promoter-dependent or promoter-independent RNAP (e.g., T7) activity; some may be affected by termination sites within the template.

Without wishing to be bound by theory, we note that a number of mechanisms have been proposed for the production of dsRNA during in vitro transcription. For example, in some cases, short RNA transcripts (e.g., about 5 to about 11nt long) may be produced when an enzyme that has initiated synthesis ceases before completion of the transcript and can then initiate transcription of the complementary strand. Alternatively or additionally, RNA folding back may lead to prolonged transcription, even generating very long (possibly double or even more times in size) transcripts. Still further alternatively or additionally, restarting transcription at certain open template structures may result in transcription to generate antisense strands. The report also shows that the template termination site may affect dsRNA production.

The invention provides, inter alia, techniques for reducing dsRNA in RNA formulations, e.g., in some embodiments, dsRNA present in RNA formulations prepared according to the present disclosure is reduced relative to dsRNA present in RNA formulations prepared, e.g., using equimolar amounts of ATP, GTP, CTP and UTP.

The level of dsRNA, and thus the reduced level thereof, may be determined using any of a variety of techniques, including but not limited to those discussed herein. For example, RNA can be spotted onto a membrane such as a nylon membrane, blocked in a suitable buffer, and detected using an antibody-based assay using an antibody specific for dsRNA, such as J2 antibody (SCICONS English and Scientific Consulting), followed by staining with a secondary antibody anti-mouse HRP antibody (Jackson ImmunoResearch) (see EP 18 717 580.7). Antibody-based detection methods are well known. RNA concentration can also be assessed using UV (e.g., nanodrop), which can also indicate whether dsRNA concentration is present. RNA integrity can be assessed using Bioanalyzer (Agilent).

The term "immunogenicity" refers to the ability of a particular substance, particularly RNA, to elicit an immune response (e.g., an innate immune response or an adaptive immune response or both) in an animal, such as a human. In other words, immunogenicity is the ability to induce humoral and/or cell-mediated immune responses. Unwanted immunogenicity includes immune responses of organisms to therapeutic substances such as drugs. This response may inactivate the therapeutic effect of the treatment and may cause adverse reactions. Without wishing to be bound by any particular theory, it is believed that the immunogenicity of many RNA preparations, particularly those produced by conventional in vitro transcription reactions, is due at least in part to the dsRNA content therein.

In some embodiments, the RNA preparations described herein (e.g., produced by the methods provided herein) are significantly less immunogenic than RNA and RNA-containing compositions transcribed from the same DNA template using previously known methods, such as using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP), or functional analogs thereof. In some embodiments, the RNA or RNA-containing composition of the invention (i.e., provided RNA formulation) is at least 5% less immunogenic than RNA or RNA-containing composition transcribed using equimolar amounts of ATP, GTP, CTP and UTP or functional analogs thereof. In some embodiments, the immunogenicity is reduced by at least 10%. In some embodiments, the immunogenicity is reduced by at least 20%. In some embodiments, the immunogenicity is reduced by at least 30%. In some embodiments, the immunogenicity is reduced by at least 40%. In some embodiments, the immunogenicity is reduced by at least 50%. In some embodiments, the immunogenicity is reduced by at least 60%. In some embodiments, the immunogenicity is reduced by at least 70%. In some embodiments, the immunogenicity is reduced by at least 80%. In some embodiments, the immunogenicity is reduced by at least 90%. In some embodiments, immunogenicity is removed or substantially removed, i.e., reduced by about 100%.

In some embodiments, the relative immunogenicity of an RNA or RNA-containing composition transcribed according to the invention and an RNA transcribed using related control methods known in the art (such as using equimolar amounts of ATP, GTP, CTP and UTP or functional analogs thereof) (i.e., the immunogenicity of the provided RNA formulation) can be determined by determining the amount of RNA provided that the provided RNA formulation elicits the same degree of result (e.g., expression of the same amount of protein) as a given amount of RNA transcribed using a control method (e.g., using equimolar amounts of ATP, GTP, CTP and UTP or functional analogs thereof) or an RNA-containing composition. In one embodiment, the relative immunogenicity of a provided RNA formulation and its corresponding counterpart transcribed using a related control method can be determined by determining the amount of a cytokine (e.g., IL-12, IFN- α, TNF- α, RANTES, MIP-1α or β, IL-6, IFN- β or IL-8) secreted in response to administration of an RNA transcribed according to the invention or a composition comprising RNA relative to the same amount of RNA transcribed using a control method or a composition comprising RNA. For example, if half of the cytokine is secreted, the RNA transcribed according to the invention or the composition comprising RNA is 50% less immunogenic than RNA transcribed using an appropriate control method or a composition comprising RNA.

By "significantly reduced immunogenicity" is meant a detectable reduction in immunogenicity. In one embodiment, the term refers to a composition that allows for the administration or repeated administration of an effective amount of RNA or RNA-containing composition without triggering a decrease in a detectable immune response. In some embodiments, the term refers to a decrease that allows repeated administration of RNA or a composition comprising RNA without eliciting an immune response sufficient to detectably reduce the expression of a peptide or protein encoded by the RNA (e.g., comprised in a composition comprising RNA). In some embodiments, the reduction allows repeated administration of the RNA or the composition comprising the RNA without eliciting an immune response sufficient to eliminate expression of the peptide or protein encoded by the RNA.

As demonstrated herein, the immunogenicity of RNA or a composition comprising RNA (i.e., RNA formulation) can be reduced by transcription of RNA using the methods according to the invention as described herein. In some embodiments, in such methods, the starting concentration of UTP or a functional analog thereof in the reaction mixture for transcription of RNA from the template is lower than the starting concentration of CTP and/or ATP or a functional analog thereof. In some embodiments, a method comprising transcribing RNA using an initial concentration of UTP or a functional analog thereof that is lower than the initial concentration of CTP and/or ATP or a functional analog thereof and supplementing the transcription reaction mixture with a composition comprising UTP or a functional analog thereof during the transcription reaction results in reduced formation of dsRNA during transcription, as compared to an appropriate control transcription reaction, such as performing the method using equimolar amounts of ATP, GTP, CTP and UTP or a functional analog thereof. Thus, in some embodiments, transcription of RNA according to the methods of the invention results in reduced immunogenicity of the RNA as compared to RNA transcribed using equimolar amounts of ATP, GTP, CTP and UTP or functional analogs thereof, as described herein. In some embodiments, transcription of RNA according to the methods of the invention results in increased RNA yield as compared to RNA transcribed using equimolar amounts of ATP, GTP, CTP and UTP or functional analogs thereof, as described herein.

In vitro transcription reaction

RNA can be synthesized in vitro, as described herein. In vitro transcription in particular allows the use of cap analogues (e.g. non-naturally occurring cap analogues) which may for example be added to the in vitro transcription reaction. Those skilled in the art will appreciate that in many embodiments, the poly (a) tail of an RNA molecule, if present, is encoded by a complementary sequence on the transcribed template (e.g., by a poly (dT) sequence in a DNA template). Alternatively or additionally, in some embodiments, capping and/or poly (a) tail addition may be achieved enzymatically after transcription, as known in the art.

Those skilled in the art will appreciate that in vitro transcription reactions generally include: (1) A template (typically DNA, typically linear) comprising a promoter that directs transcription of the sequence of interest; (2) Ribonucleoside triphosphates (which may be natural compounds or analogues); (3) a buffer system (typically comprising magnesium ions); and (4) RNA polymerase. Because ofThus, according to the present invention, the RNA transcription reaction generally comprises: (1) Templates (e.g., DNA, typically linear) that may contain promoters that direct transcription of the sequences of interest; (2) Ribonucleoside triphosphates (natural or functional analogs thereof); (3) Buffer systems, such as the buffers described herein, are selected, for example, according to the RNA polymerase used; and (4) RNA polymerase. In some embodiments, the transcription reaction mixture may further comprise an Rnase inhibitor. In some embodiments, the transcription reaction mixture can also include pyrophosphatase (e.g., inorganic pyrophosphatase). In some embodiments, the transcription reaction mixture may also include one or more salts (e.g., monovalent salts and/or divalent salts, such as Li-containing ⁺ 、Na ⁺ 、K ⁺ 、NH ⁴⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ (ii) a salt thereof), a reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, etc.), spermidine, or a combination thereof. In some embodiments, certain reaction components are added in a particular order (e.g., pyrophosphatase and polymerase are added last). In some embodiments, the agitation rate is increased after the addition of a particular reaction component (e.g., pyrophosphatase, polymerase).

Those skilled in the art will appreciate that, particularly for large scale (e.g., greater than about 50ug or more typically greater than about 100ug of RNA per reaction, or in the range of about 120ug to about 180ug of RNA per ug of template), it is desirable to reduce variation in vitro transcription reactions. For example, in many embodiments, it may be desirable to control the concentration of monovalent or divalent cations and/or the reaction temperature. In some embodiments, the cation in the reaction mixture according to the invention is Li ⁺ 、Na ⁺ 、K ⁺ 、NH ⁴⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ . For example, in many embodiments, it may be desirable to control Mg ²⁺ Concentration and/or reaction temperature of (c). In some embodiments, mg is reduced ²⁺ And/or increasing the temperature (see, e.g., mu et al, nuc Acids Res. 10:5239).

In some embodiments, the present disclosure provides, inter alia, techniques for large-scale manufacturing of pharmaceutical grade compositions or formulations comprising RNA, e.g., at a large-scale throughput of at least 10g RNA (including, e.g., at least 15g RNA, at least 20g RNA, at least 25g RNA, at least 30g RNA, at least 35g RNA, at least 40g RNA, at least 45g RNA, at least 50g RNA, at least 55g RNA, at least 60g RNA, at least 70g RNA, at least 80g RNA, at least 90g RNA, at least 100g RNA, at least 150g RNA, at least 200g RNA, or more). In some embodiments, such methods described herein can be used to produce a mass throughput of about 10g to about 300g RNA, about 10g to about 200g RNA, about 10g to about 100g RNA, about 30g to about 60g RNA, or about 50g to 300g RNA. In some embodiments, such methods described herein can be used for large scale manufacturing that produces at least 1g RNA/hr or more (such as at least 1.5g RNA/hr, including, for example, at least 2g RNA/hr, at least 2.5g RNA/hr, at least 3g RNA/hr, at least 3.5g RNA/hr, at least 4g RNA/hr, at least 4.5g RNA/hr, at least 5g RNA/hr, at least 5.5g RNA/hr, at least 6g RNA/hr, at least 6.5g RNA/hr, at least 7g RNA/hr, at least 7.5g RNA/hr, at least 8g RNA/hr, at least 8.5g RNA/hr, at least 9g RNA/hr, at least 10g RNA/hr, or more). In some embodiments, the large-scale manufacturing methods described herein can reach a capacity of 15g RNA/hr to 20g RNA/hr (e.g., about 17 g/hr).

Those skilled in the art will appreciate that the term "polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymer molecule from monomeric building blocks. An "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. An "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. A "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, DNA polymerases synthesize DNA molecules based on a template nucleic acid, which is typically a DNA molecule. In general, RNA polymerases synthesize RNA molecules based on a template nucleic acid, which is either a DNA molecule (in the case where the RNA polymerase is a DNA-dependent RNA polymerase DdRP) or an RNA molecule (in the case where the RNA polymerase is an RNA-dependent RNA polymerase RdRP).

Various RNA polymerases suitable for transcription reactions are known in the art, including but not limited to DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or variants or functional domains thereof). Naturally catalyzed RNA-dependent RNA polymerase is typically encoded by all RNA viruses except retroviruses. A typical representation of viruses encoding RNA-dependent RNA polymerase is alphavirus. The skilled artisan will appreciate that the RNA polymerase used herein may be a recombinant RNA polymerase and/or a purified RNA polymerase, i.e., not as part of a cell extract that contains other components in addition to the RNA polymerase. In some embodiments, the RNA polymerase that can be used for commercial scale transcription is T7 RNA polymerase. In some embodiments, inorganic pyrophosphatase may be added to increase the yield of transcription reactions (e.g., catalyzed by T7 RNA polymerase in some embodiments).

The present disclosure particularly identifies that limiting nucleotides can reduce dsRNA formation in an in vitro reaction. The present disclosure demonstrates in particular that limiting UTP in vitro transcription reactions can reduce dsRNA formation, and can be particularly useful for producing transcripts that can include polyA sequences (such as polyA tails). Without wishing to be bound by theory, we propose that the observed reduction in dsRNA production may be due to a reduction in reverse transcription (e.g., initiation upon hybridization to a polyA sequence (such as a polyA tail)).

Those of skill in the art will understand that "restriction of UTP" as used herein means a constraint on the level of nucleotides functionally paired with the a residues in the template such that they are used in templated synthesis (i.e., in an in vitro transcription reaction) and incorporated into the resulting strand; such nucleotides are referred to herein as "UTP or a functional analogue thereof"). In some embodiments, the functional analog is also translatable, typically as a "U".

Thus, the present disclosure provides, inter alia, certain in vitro transcription techniques in which the concentration of UTP (and/or functional analogs thereof) is limited (e.g., lower than the concentration of CTP and/or ATP or functional analogs thereof). In some embodiments, UTP is limited at the beginning of the reaction. In some embodiments, UTP is limited throughout the reaction.

For example, the present disclosure provides in vitro transcription reactions in which UTP is limited (e.g., the concentration of UTP and its functional analogs is lower than the concentration of one or more other nucleotides, i.e., adenosine Triphosphate (ATP) and its functional analogs, guanosine Triphosphate (GTP) and its functional analogs, and Cytidine Triphosphate (CTP) and its functional analogs).

Furthermore, the present disclosure provides, inter alia, techniques in which UTP is limited in an initial in vitro transcription reaction as described herein. In some embodiments, the present disclosure provides, inter alia, techniques in which an initial in vitro transcription reaction with UTP limitation is supplemented with UTP or a functional analog thereof over time; in some such embodiments, such replenishment is by one or more discrete feed events. In some embodiments, such supplementation may be performed by a continuous process (e.g., in some embodiments, where the rate of UTP supplementation is comparable to the rate of UTP consumption during the transcription reaction). In some embodiments, UTP is limited during supplementation, e.g., at a concentration such that, upon such supplementation, UTP or a functional analog thereof is present in the reaction at a concentration that is lower than the concentration of one or more (and in some embodiments, all) of ATP or a functional analog thereof, GTP or a functional analog thereof, and/or CTP or a functional analog thereof.

In some embodiments, the in vitro transcription reaction is supplemented with a plurality of nucleotides (e.g., with UTP or a functional analog thereof, and also with one or more other nucleotides (e.g., ATP or a functional analog thereof, GTP or a functional analog thereof, and/or CTP or a functional analog thereof)).

In some embodiments, both UTP and GTP are limited in the initial reaction and/or during supplementation.

RNA products

The present disclosure provides techniques that can be used to produce RNA products (i.e., formulations for making particular RNA products).

In certain embodiments of the present disclosure, the RNA produced is, for example, messenger RNA (mRNA) that relates to an RNA transcript encoding a peptide or protein. As is known in the art, in many cases, mRNA may contain a 5 'untranslated region (5' utr), a peptide coding region, and a 3 'untranslated region (3' utr).

In some embodiments, the RNA product is produced by in vitro transcription. In some embodiments, the mRNA product is produced by in vitro transcription, for example, using a DNA template (where DNA refers to a nucleic acid containing deoxyribonucleotides). In some embodiments, RNA synthesis may also occur within a cell or other system.

In some embodiments, the RNA product is in vitro transcribed RNA (IVT-RNA) and is obtainable by in vitro transcription of an appropriate DNA template.

In certain embodiments, the DNA template is a linear molecule. In certain embodiments, the DNA template is a circular molecule. In general, DNA can be obtained or generated using methods known in the art, including, for example, gene synthesis, recombinant DNA techniques, or combinations thereof. In some embodiments, the DNA template comprises a nucleotide sequence encoding a transcribed region of interest (e.g., encoding an RNA, such as the RNAs described herein) and a promoter sequence selected for recognition by an RNA polymerase for in vitro transcription. The promoter used to control transcription may be any promoter for any RNA polymerase. Various RNA polymerases are known in the art, and exemplary polymerases are disclosed herein. Those of skill in the art will readily appreciate upon reading this disclosure that the RNA polymerase used herein may be a recombinant RNA polymerase and/or a purified RNA polymerase, i.e., not as part of a cell extract that contains other components in addition to the RNA polymerase. Those skilled in the art will recognize the appropriate promoter sequence for the selected RNA polymerase. In some embodiments, the DNA template may comprise a promoter sequence for a T7 RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning a nucleic acid such as cDNA and introducing it into an appropriate vector for in vitro transcription. Techniques for introducing nucleic acid sequences into vectors are well known in the art, such as cold fusion cloning and other techniques. cDNA can be obtained by reverse transcription of RNA.

In some embodiments, the RNA suitable for use in the techniques described herein is single-stranded RNA (e.g., mRNA as described herein). In some embodiments, the single-stranded RNA is non-coding RNA in that its nucleotide sequence does not include an open reading frame (or its complement). In some embodiments, the single stranded RNA has a nucleotide sequence encoding a polypeptide (or a complement of a sequence encoding the polypeptide) as described herein.

In some embodiments, the techniques described herein may be particularly useful for synthesizing single-stranded RNAs having a length of at least 500 ribonucleotides (such as at least 600 ribonucleotides, at least 700 ribonucleotides, at least 800 ribonucleotides, at least 900 ribonucleotides, at least 1000 ribonucleotides, at least 1250 ribonucleotides, at least 1500 ribonucleotides, at least 1750 ribonucleotides, at least 2000 ribonucleotides, at least 2500 ribonucleotides, at least 3000 ribonucleotides, at least 3500 ribonucleotides, at least 4000 ribonucleotides, at least 4500 ribonucleotides, at least 5000 ribonucleotides, at least 6000 ribonucleotides, at least 7000 ribonucleotides, at least 8000 ribonucleotides, at least 9000 ribonucleotides, at least 10000 ribonucleotides, at least 11000 ribonucleotides, at least 12000 ribonucleotides, or longer). In some embodiments, the techniques described herein may be particularly useful for synthesizing single stranded RNAs having a length of about 1000 ribonucleotides to 5000 ribonucleotides.

In some embodiments, the related RNA includes a polypeptide-encoding portion. In some particular embodiments, such a moiety may encode a polypeptide that is or comprises an antigen (or epitope thereof), a cytokine, an enzyme, or the like. In some embodiments, the encoded polypeptide may be or include one or more tumor-associated neoantigens or neoepitopes. In some embodiments, the encoded polypeptide may be or include an antigen (or epitope thereof) of an infectious agent (e.g., bacteria, fungi, viruses, etc.). In certain embodiments, the encoded polypeptide may be a variant of the wild-type polypeptide.

The present disclosure specifically exemplifies the manufacture of RNAs encoding viral antigens (and/or epitopes thereof) (e.g., coronavirus antigens and/or epitopes). For example, in some embodiments, the present disclosure exemplifies the production of single stranded RNA whose nucleotide sequence encodes a coronavirus polypeptide or variant thereof. In some embodiments, the single stranded RNA comprises a nucleotide sequence encoding a pre-fusion coronavirus spike protein, e.g., as described in WO 2018081318 (the entire contents of which are incorporated herein by reference for the purposes described herein).

In some embodiments, the single stranded RNA comprises a nucleotide sequence encoding a SARS-CoV-2 polypeptide, including, for example, spike (S) protein, nucleocapsid (N) protein, envelope (E) protein, and membrane (M) protein, or immunogenic fragments thereof. In some embodiments, the single stranded RNA comprises a nucleotide sequence encoding a SARS-CoV-2S polypeptide or an immunogenic fragment thereof (e.g., the receptor binding domain of the S protein). In some embodiments, such SARS-CoV-2S polypeptide or immunogenic fragment thereof can be a mutein. In some embodiments, the RNA used in accordance with the present disclosure encodes SARS-CoV-2 spike protein having the K986P and V978P mutations.

In some embodiments, single-stranded RNA (e.g., mRNA as described herein) can comprise a secretion signal encoding region (e.g., a secretion signal encoding region that allows the encoded target entity to be secreted after translation by the cell). In some embodiments, such secretion signal encoding regions may be or comprise non-human secretion signals. In some embodiments, such a secretion signal encoding region may be or comprise a human secretion signal.

In some embodiments, single-stranded RNA (e.g., mRNA as described herein) can comprise at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, 3 'untranslated regions (UTRs), 5' UTRs, co-transcribed capped cap structures for mRNA, poly adenine (polyA) tails, and any combination thereof.

UTR (5 'UTR and/or 3' UTR): in some embodiments, the single stranded RNA may comprise a nucleotide sequence encoding a 5'utr of interest and/or a 3' utr of interest. Those skilled in the art will appreciate that untranslated regions of mRNA sequences (e.g., 3 'utrs and/or 5' utrs) may contribute to mRNA stability, mRNA localization, and/or translation efficiency. In some embodiments, the untranslated region (UTR) may be present 5 '(upstream) of the open reading frame (5' UTR) and/or 3 '(downstream) of the open reading frame (3' UTR).

In some embodiments, the single stranded RNA can comprise a 5'utr nucleotide sequence and/or a 3' utr nucleotide sequence. In some embodiments, such 5'utr sequences may be operably linked to 3' of a coding sequence (e.g., comprising one or more coding regions). Additionally or alternatively, in some embodiments, the 3'utr sequence may be operably linked to 5' of the coding sequence (e.g., comprising one or more coding regions). In some embodiments, according to the invention, the 5 '-and/or 3' -untranslated regions may be functionally linked to the open reading frame such that these regions are associated with the open reading frame in such a way: such that the stability and/or translation efficiency of the RNA comprising said open reading frame is increased.

In some embodiments, the 5 'and 3' utr sequences included in the single stranded RNA may consist of or comprise naturally occurring or endogenous 5 'and 3' utr sequences of the open reading frame of the gene of interest. Alternatively, in some embodiments, the 5 'and/or 3' utr sequences included in the single stranded RNA are not endogenous to the coding sequence (e.g., comprise one or more coding regions); in some such embodiments, such 5 'and/or 3' utr sequences may be used to modify the stability and/or translation efficiency of transcribed RNA sequences. For example, the skilled artisan will appreciate that AU-rich elements in the 3' UTR sequence may reduce the stability of mRNA. Thus, as will be appreciated by the skilled artisan, the 3 'and/or 5' UTRs may be selected or designed to increase the stability of transcribed RNA based on the characteristics of UTRs well known in the art.

For example, one of skill in the art will appreciate that in some embodiments a Kozak sequence consisting of or comprising the open reading frame sequence of a gene or nucleotide sequence of interest may be selected and used as the nucleotide sequence encoding the 5' utr. As the skilled artisan will appreciate, kozak sequences are known to increase the efficiency of translation of some RNA transcripts, but not all RNAs are necessary to achieve efficient translation. In some embodiments, the single stranded RNA may comprise a nucleotide sequence encoding a 5' utr derived from an RNA virus whose RNA genome is stable in the cell. In some embodiments, various modified ribonucleotides (e.g., as described herein) can be used in the 3 'and/or 5' utr, e.g., to prevent exonuclease degradation of transcribed RNA sequences.

As known to those skilled in the art, a Kozak sequence is a sequence originally described by Kozak, (1987), nucleic Acids Res., 15:8125-8148. The Kozak sequence on an RNA molecule, such as an mRNA molecule, is recognized by the ribosome as a translation initiation site. Typically, the Kozak sequence comprises an AUG start codon followed by a highly conserved G nucleotide: AUGG. In particular, it is described that the Kozak sequence can be identified by (gcc) gccRccAUGG as follows: (i) Lowercase letters denote the most common bases at positions where the bases can still vary; (ii) Capital letters indicate highly conserved bases (e.g., "AUGG"); (iii) R "represents purine (adenine or guanine); (iv) the sequence in brackets ((gcc)) is of unknown significance; (v) The underlined AUG base triplet represents the start codon.

In some embodiments, the RNA produced according to the invention comprises

(1)5'UTR，

(2) For example, an open reading frame encoding a peptide or protein of interest, and/or

(3)3'UTR。

In some embodiments, such 5'utr sequences may be operably linked to 3' of a coding sequence (e.g., comprising one or more coding regions). Additionally or alternatively, in some embodiments, the 3'utr sequence may be operably linked to 5' of the coding sequence (e.g., comprising one or more coding regions).

In some embodiments, the 5' utr included in the single stranded RNA may be derived from human α -globin mRNA in combination with a Kozak region.

In some embodiments, the single stranded RNA can comprise one or more 3' utrs. For example, in some embodiments, a single-stranded RNA can comprise two copies of a 3' -UTR derived from a globin mRNA, such as a 2-globin, a 1-globin, β -globin (e.g., human β -globin) mRNA. In some embodiments, two copies of the 3' UTR derived from human β -globin mRNA may be used, e.g., in some embodiments, it may be placed between the coding sequence of single stranded RNA and the poly (A) tail to increase the protein expression level and/or extend the persistence of the mRNA. In some embodiments, the 3'utr included in the single stranded RNA may be or comprise one or more (e.g., 1, 2, 3, or more) of the 3' utr sequences disclosed in WO 2017/060314 (the entire contents of which are incorporated herein by reference for the purposes of this disclosure). In some embodiments, the 3' -UTR may be a combination of at least two sequence elements (FI elements) derived from a "split amino terminal enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I). These were identified by an ex vivo selection procedure for sequences that confer RNA stability and increase total protein expression (see WO 2017/060314, which is incorporated herein by reference).

Two consecutive identical copies of the human beta-globin 3'UTR, in particular the human beta-globin 3' UTR, contribute to high transcript stability and translation efficiency (Holtkamp et al, (2006), blood, 108:4009-4017). Thus, in some embodiments, a replicase construct according to the invention comprises two consecutive identical copies of the human β -globin 3' utr. Thus, it comprises in the 5'→3' direction: (a) optionally a 5' utr; (b) an open reading frame; (c) a 3' UTR; the 3' utr comprises two contiguous identical copies of a human β -globin 3' utr, a fragment thereof, or a variant of a human β -globin 3' utr or a fragment thereof.

In some embodiments, the RNA according to the invention comprises a 3' utr that is active to increase translation efficiency and/or stability but is not a human β -globin 3' utr, a fragment thereof, or a variant of a human β -globin 3' utr or fragment thereof.

polyA tail: in some embodiments, the single stranded RNA may comprise a polyA tail. A polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which may vary in length (e.g., at least 5 adenine nucleotides) and may be up to several hundred adenosine nucleotides. The polyA tail may be transcribed from a template (e.g., a DNA template), or may be added enzymatically after the transcription reaction. In some embodiments, the polyA tail is a nucleotide sequence comprising at least 30 or more adenosine nucleotides (including, e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more adenosine nucleotides). In some embodiments, the polyA tail is or comprises a polyA homopolymer tail. In some embodiments, the polyA tail may comprise one or more modified adenosine nucleosides including, but not limited to, cordycepin and 8-azaadenosine. In some embodiments, the polyA tail may comprise one or more non-adenosine nucleotides. In some embodiments, the polyA tail may be or comprise a disrupted or modified polyA tail as described in WO 2016/005324 (the entire contents of which are incorporated herein by reference for the purposes of this description). For example, in some embodiments, the polyA tail included in a single-stranded RNA described herein can be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 consecutive a nucleotides, which is 5' of the linker sequence; and a second sequence of at least 20 consecutive a nucleotides, which is 3' of the linker sequence. In some embodiments, the modified polyA sequence may comprise: a linker sequence comprising at least ten nucleotides (e.g., U, G and/or C nucleotides); a first sequence of at least 30 consecutive a nucleotides, which is 5' of the linker sequence; and a second sequence of at least 70 consecutive a nucleotides, which is 3' of the linker sequence.

In some embodiments, the poly (a) tail comprises, consists essentially of, or consists of at least 20 nucleotides, in some embodiments at least 26 nucleotides, in some embodiments at least 40 nucleotides, in some embodiments at least 80 nucleotides, in some embodiments at least 100 nucleotides and in some embodiments at most 500 nucleotides, in some embodiments at most 400 nucleotides, in some embodiments at most 300 nucleotides, in some embodiments at most 200 nucleotides, in some embodiments at most 150 and especially about 120 a nucleotides. In some embodiments, the poly (a) tail is a nucleotide sequence comprising at least 30 or more adenosine nucleotides (including, e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more adenosine nucleotides). Herein, "consisting essentially of … …" means that most of the nucleotides in the poly (a) tail (typically at least 50%, and in some embodiments at least 75% by number of nucleotides in the "poly (a) tail) are a nucleotides (adenylates), but the remaining nucleotides are allowed to be nucleotides other than a nucleotides, such as U nucleotides (uridylates), G nucleotides (guanylate), C nucleotides (cytidylates). Herein, "consisting of … …" means that all nucleotides in the poly (a) tail (i.e., 100% by number of nucleotides in the "poly (a) tail") are a nucleotides. The term "a nucleotide" or "a" refers to an adenylate.

In fact, a 3' poly (A) tail of about 120A nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells as well as on protein levels translated from an open reading frame present upstream (5 ') of the 3' poly (A) tail (Holtkamp et al, (2006), blood, 108:4009-4017).

According to the present invention, in some embodiments, a 3' poly (A) tail is attached during RNA transcription, i.e., during the preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylates) in the strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly (A) tail is referred to as the poly (A) cassette.

5' cap: in some embodiments, an RNA product (e.g., single-stranded RNA) prepared as described herein can comprise a 5' cap that can be incorporated into such single-stranded RNA during transcription, or that binds to such RNA post-transcriptionally. In some embodiments, the RNA can comprise a 5' cap structure for co-transcriptional capping of the mRNA. Examples of cap structures for co-transcribing capping are known in the art, including for example as described in WO 2017/053297 (the entire contents of which are incorporated herein by reference for the purposes described herein). In some embodiments, the 5' cap included in the RNA products described herein is or is a bag Contains cap1 structure. For example, in some embodiments, the cap1 structure may be or comprise m7G (5 ') ppp (5 ') (2 ' ome) pG, also referred to as m ₂ ^7,3'- ^O Gppp(m ₁ ^2'-O )ApG。

In some embodiments, an RNA product (e.g., single-stranded RNA) produced as described herein may comprise at least one modified ribonucleotide, e.g., in some embodiments to increase the stability of such RNA product and/or reduce the cytotoxicity of such RNA product. For example, in some embodiments, at least one of the A, U, C and G ribonucleotides of the single-stranded RNA can be replaced with a modified ribonucleotide. For example, in some embodiments, some or all of the cytidine residues present in the single-stranded RNA can be replaced with a modified cytidine, which in some embodiments can be, for example, 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all of the uridine residues present in the single stranded RNA may be replaced by a modified uridine, which in some embodiments may be, for example, a pseudouridine, such as 1-methyl pseudouridine. In some embodiments, all uridine residues present in single stranded RNA are replaced with pseudouridine (e.g., 1-methyl pseudouridine).

DNA template

Those of ordinary skill in the art will appreciate that DNA templates are commonly used to guide the synthesis of RNA (e.g., single stranded RNA). In some embodiments, the DNA template is a linear DNA molecule. In some embodiments, the DNA template is a circular DNA molecule. DNA may be obtained or generated using methods known in the art, including, for example, gene synthesis, recombinant DNA techniques, or combinations thereof. In some embodiments, the DNA template comprises a nucleotide sequence encoding a transcribed region of interest (e.g., encoding an RNA described herein) and a promoter sequence selected for recognition by an RNA polymerase for in vitro transcription. Various RNA polymerases are known in the art, including, for example, DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or variants or functional domains thereof). The skilled artisan will readily appreciate that the RNA polymerase used herein may be a recombinant RNA polymerase and/or a purified RNA polymerase, i.e., not as part of a cell extract that contains other components in addition to the RNA polymerase. Those skilled in the art will recognize the appropriate promoter sequence for the selected RNA polymerase. In some embodiments, the DNA template may comprise a promoter sequence for a T7 RNA polymerase.

In some embodiments, the DNA template comprises a nucleotide sequence encoding an RNA described herein (e.g., comprises a nucleotide sequence encoding an antigen of interest and optionally comprises one or more nucleotide sequences encoding a characteristic element of an RNA described herein (including, e.g., a polyA tail, 3'utr, and/or 5' utr, etc.). In some embodiments, such coding sequences may be generated by genetic synthesis. In some embodiments, such coding sequences may be inserted into vectors by cold fusion cloning.

In some embodiments, the DNA template may further comprise one or more of a recognition sequence for a suitable restriction endonuclease (e.g., for linearization), a suitable resistance gene, and/or a suitable origin of replication. In some embodiments, the DNA template may further comprise a recognition sequence for a suitable restriction endonuclease (e.g., for linearization, such as but not limited to a class II restriction endonuclease), a suitable resistance gene (e.g., but not limited to a kanamycin resistance gene), and a suitable origin of replication.

In some embodiments, the DNA template may be amplified or have been amplified, for example, from plasmid DNA via Polymerase Chain Reaction (PCR). In some embodiments, plasmid DNA may be obtained, for example, from bacterial cells (e.g., escherichia coli), followed by an endotoxin-free and animal product-free plasmid isolation procedure.

Ribonucleotides

Ribonucleotides for in vitro transcription may comprise at least two or more (e.g., at least three or more, at least four or more, at least five or more, at least six or more) different types of ribonucleotides, each type having a different nucleoside. Ribonucleotides for in vitro transcription may comprise unmodified and/or modified ribonucleotides. Unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). In some embodiments, all four types of unmodified ribonucleotides can be used for in vitro transcription.

In some embodiments, at least one type of ribonucleotide included in vitro transcription is a modified ribonucleotide. Modified ribonucleotides can include one or more modifications including, but not limited to, for example, (a) terminal modifications, such as 5 'terminal modifications (e.g., phosphorylations, dephosphorations, conjugates, reverse linkages, etc.), 3' terminal modifications (e.g., conjugates, reverse linkages, etc.), base modifications, (b) base modifications, such as base substitutions with modified bases, stabilized bases, destabilized bases, or base pairing with all components of the amplified partner, or conjugated bases, (c) sugar modifications (e.g., at the 2 'position or the 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages. To the extent that such modifications interfere with translation (e.g., result in 50% or more reduction in translation relative to the absence of modification-e.g., as characterized using rabbit reticulocyte in vitro translation assays), such modified ribonucleotides are not suitable for use in the techniques described herein in some embodiments.

In some embodiments, one or more modified nucleosides may be used, such as one or more functional analogs of one or more of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP). When a functional analogue of either ATP, GTP, CTP or UTP is used in the method according to the invention, the resulting RNA molecule will comprise these functional analogues instead of ATP, GTP, CTP or UTP, respectively.

In some embodiments, the modified ribonucleotides may have at least one nucleoside ("base") modification or substitution. Various nucleoside modifications or substitutions are known in the art; those skilled in the art will appreciate that modified nucleosides include, for example and without limitation, synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, gourmetin (nubularine), isoguanosine, tubercidin (tubercidin), 2- (halo) adenine, 2- (alkyl) adenine, 2- (propyl) adenine, 2- (amino) adenine, 2- (aminoalkyl) adenine, 2- (aminopropyl) adenine, 2- (methylthio) -N6- (isopentenyl) adenine, 6- (alkyl) adenine, 6- (methyl) adenine, 7- (deazaadenine, 8- (alkenyl) adenine, 8- (alkyl) adenine, 8- (alkynyl) adenine, 8- (amino) adenine, 8- (halo) adenine, 8- (hydroxy) adenine, 8- (thioalkyl) adenine, 8- (thiol) adenine, N6- (isopentyl) adenine, N6- (methyl) adenine, N6- (dimethyl) adenine, 2- (alkyl) adenine, 2- (methyl) guanine, 7- (alkyl) guanine, 7- (methyl) guanine 8- (alkyl) guanine, 8- (alkenyl) guanine, 8- (alkynyl) guanine, 8- (amino) guanine, 8- (halo) guanine, 8- (hydroxy) guanine, 8- (thioalkyl) guanine, 8- (thiol) guanine, N- (methyl) guanine, 2- (thio) cytosine, 3- (deaza) -5- (aza) cytosine, 3- (alkyl) cytosine, 3- (methyl) cytosine, 5- (alkyl) cytosine, 5- (alkynyl) cytosine, 5- (halo) cytosine, 5- (methyl) cytosine, 5- (propynyl) cytosine, 5- (trifluoromethyl) cytosine, 6- (azo) cytosine, N4- (acetyl) cytosine, 3- (3-amino-3-carboxypropyl) uracil, 2- (thio) uracil, 5- (methyl) -2- (thio) uracil, 5- (methylaminomethyl) -2- (thio) uracil, 4- (thio) uracil, 5- (methyl) -4- (thio) uracil, 5- (methylaminomethyl) -4- (thio) uracil, 5- (methyl) -2,4- (dithio) uracil, 5- (methylaminomethyl) -2,4- (dithio) uracil, 5- (2-aminopropyl) uracil, 5- (alkyl) uracil, 5- (alkynyl) uracil, 5- (allylamino) uracil, 5- (amino allyl) uracil, 5- (aminoalkyl) uracil, 5- (guanidyl) uracil, 5- (l, 3-diazole-l-alkyl) uracil, 5- (cyanoalkyl) uracil, 5- (dialkylaminoalkyl) uracil, 5- (dimethylaminoalkyl) uracil, 5- (halo) uracil, 5- (methoxy) uracil, uracil-5-glycolate, 5- (methoxycarbonylmethyl) -2- (thio) uracil, 5- (methoxycarbonyl-methyl) uracil, 5- (propynyl) uracil, 5- (trifluoromethyl) uracil, 6- (azo) uracil, 2- (3-methyl) uracil, 3- (chloro) uracil, 3-methyl) uracil, 2- (pseudouracil, 2-methyl) uracil, 4- (thio) pseudouracil, 2,4- (dithio) pseudouracil, 5- (alkyl) pseudouracil, 5- (methyl) pseudouracil, 5- (alkyl) -2- (thio) pseudouracil, 5- (methyl) -2- (thio) pseudouracil, 5- (alkyl) -4- (thio) pseudouracil, 5- (methyl) -4- (thio) pseudouracil, 5- (alkyl) -2,4- (dithio) pseudouracil, 5- (methyl) -2,4- (dithio) pseudouracil, 1-substituted pseudouracil (e.g., 1-methyl-pseudouridine), C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-iodouridine, C5-propynyl-uridine, 1-substituted 2 (thio) -pseudouracil, 1-substituted 4- (thio) pseudouracil, 1-substituted 2,4- (dithio) pseudouracil, 1- (pseudocarbonyl) -amino-ethyl-uracil, 1- (amino-carbonyl) -amino-ethyl-uracil, pseudocarbonyl-amino-ethyl-uracil, amino-ethyl-2- (thio) pseudouracil, 1- (aminocarbonylethyl alkenyl) -2,4- (dithio) pseudouracil, 1- (aminoalkylaminocarbonylethyl alkenyl) -pseudouracil, 1- (aminoalkylamino-carbonylathylalkenyl) -2 (thio) -pseudouracil, 1- (aminoalkylaminocarbonylethyl alkenyl) -4- (thio) pseudouracil, 1- (aminoalkylaminocarbonylethyl alkenyl) -2,4- (dithio) pseudouracil, l,3- (diaza) -2- (oxo) -benzoxazin-l-yl, l- (aza) -2- (thio) -3- (aza) -benzoxazin-l-yl, l,3- (diaza) -2- (oxo) -phenothiazin-l-yl, l- (aza) -2- (thio) -3- (aza) -phenothiazin-l-yl, 7-substituted 1,3- (diaza) -2- (oxo) -benzoxazin-l-yl, 7-substituted l- (aza) -2- (aza) -3- (aza) -benzoxazin-l-yl, 7-substituted l,3- (aza) -benzoxazin-l-yl, 3- (aza) -phenothiazin-l-yl, 7-substituted l- (aza) -2- (thio) -3- (aza) -phenothiazin-l-yl, 7- (aminoalkylhydroxy) -l,3- (diaza) -2- (oxo) -benzoxazin-l-yl, - (aminoalkylhydroxy) -l- (aza) -2- (thio) -3- (aza) -benzoxazin-l-yl, 7- (aminoalkylhydroxy) -l,3- (diaza) -2- (oxo) -phenothiazin-l-yl, 7- (aminoalkylhydroxy) -l- (aza) -2- (thio) -3- (aza) -phenothiazin-l-yl, 7- (guanidyl hydroxy) -l,3- (diaza) -2- (oxo) -benzoxazin-l-yl, 7- (guanidyl alkyl hydroxy) -l- (aza) -2- (thio) -3- (aza) -benzoxazin-1-yl, 7- (guanidyl-hydroxy) -1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7- (guanidyl alkyl hydroxy) -l- (aza) -2- (thio) -3- (aza) -phenothiazin-l-yl, 1,3,5- (triaza) -2,6- (dioxa) -naphthalene, inosine, xanthine, hypoxanthine, gourmet powder mushroom, tubercidin, isoguanosine, inosine, 2-aza-inosine, 7-deaza-inosine, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolo pyrimidine, 3- (methyl) isoquinolone, 5- (methyl) isoquinolone, 3- (methyl) -7- (propynyl) isoquinolone 7- (aza) indolyl, 6- (methyl) -7- (aza) indolyl, imidazopyridinyl, 9- (methyl) -imidazopyridinyl, pyrrolopyridinyl, isoquinolinyl, 7- (propynyl) isoquinolinyl, propynyl-7- (aza) indolyl, 2,4,5- (trimethyl) phenyl, 4- (methyl) indolyl, 4,6- (dimethyl) indolyl, phenyl, naphthyl, anthracenyl, phenanthryl, pyrenyl, stilbene, tetracenyl, fused pentacenyl, difluoromethyl phenyl, 4- (fluoro) -6- (methyl) benzimidazole, 4- (methyl) benzimidazole, 6- (azo) thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6- (aza) pyrimidine, 2- (amino) purine, 2,6- (diamino) purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, 06-substituted purine, substituted 1,2, 4-triazole, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyrido-7-amino-pyrimidin-3-one-yl, 2-oxo-pyridopyrimidin-3-yl or any O-alkylated or N-alkylated derivative thereof.

In some embodiments, modified nucleotides used in the IVT systems and/or methods described herein can disrupt the binding of RNA to one or more mammalian (e.g., human) endogenous RNA sensors (e.g., innate immune RNA sensors), including, for example, but not limited to, toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid inducible gene I (RIG-I), melanoma differentiation associated gene 5 (MDA 5), protein Kinase R (PKR), 2'-5' oligoadenylate synthetase (OAS), and genetic and physiological laboratory protein 2 (LGP 2), and combinations thereof. In some embodiments, such modified ribonucleotides may include modifications as described in US 9,334,328 (the contents of which are incorporated herein by reference in their entirety for the purposes described herein). It is generally desirable that the modified nucleoside is translatable in the host cell (e.g., the presence of the modified nucleoside does not prevent translation of the RNA sequence into the corresponding protein sequence). The effect of the modified nucleotide on translation can be determined by one of ordinary skill in the art using, for example, a rabbit reticulocyte lysate translation assay.

In some embodiments, modified ribonucleotides may include modified internucleoside linkages. Various such modified internucleoside linkages are known in the art; those skilled in the art will appreciate that non-limiting examples of modified internucleoside linkages useful in the techniques provided herein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -phosphoramidates and aminoalkyl phosphoramidates), thiocarbonyl phosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reverse polarity in which adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5 '-2'. Also included are various salts, mixed salts and free acid forms. Modified internucleoside linkages not including phosphorus atoms therein may have internucleoside linkages formed from short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar moiety of a nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioacetylacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and others with mixed N, O, S and CH2 component parts.

In some embodiments, the modified ribonucleotides may include one or more substituted sugar moieties. Various such modified sugar moieties are known in the art; those skilled in the art will appreciate that in some embodiments, the sugar portion of the ribonucleotide may comprise one of the following at the 2' position: h (deoxyribose); OH (ribose); f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted. In some embodiments, the sugar portion of the ribonucleotide can include 2 'methoxyethoxy (2' -O-CH) ₂ CH ₂ OCH ₃ Also known as 2'-O- (2-methoxyethyl) or 2' -MOE), 2 '-dimethylaminooxyethoxy (i.e., O (CH 2) 2ON (CH 3) 2 group, also known as 2' -DMAOE) and 2 '-dimethylaminoethoxyethoxy (also known in the art as 2' O-dimethylaminoethoxyethyl or 2 '-DMAEOE) (i.e., 2' -O-CH) ₂ -O-CH ₂ -N(CH ₂ ) ₂ ) 2 '-methoxy (2' -OCH) ₃ ) 2 '-aminopropoxy (2' -OCH) ₂ CH ₂ CH ₂ NH ₂ ) And 2 '-fluoro (2' -F). Similar modifications can also be made at other positions (e.g., the 3 'position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked nucleotide, and the 5 'position of the 5' terminal nucleotide).

In some embodiments, the mixture of ribonucleotides useful in an in vitro transcription reaction may comprise UTP or a functional analog thereof in combination with at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and GTP or a functional analog thereof. In some embodiments, the functional analog of UTP is or comprises N1-methyl pseudouridine-5' triphosphate (m 1 ψTP). As described herein, the present disclosure provides, inter alia, techniques in which UTP is limited as described herein in an initial in vitro transcription reaction (e.g., at a concentration that is lower than the concentration of ATP or a functional analog thereof, GTP or a functional analog thereof, and/or CTP or one or more (and in some embodiments all) of the functional analogs thereof). In some embodiments, the present disclosure provides, inter alia, techniques in which an initial in vitro transcription reaction with UTP limitation is supplemented with UTP or a functional analog thereof over time; in some such embodiments, such replenishment is by one or more discrete feed events. In some embodiments, such supplementation may be performed by a continuous process (e.g., in some embodiments, where the rate of UTP supplementation is comparable to the rate of UTP consumption during the transcription reaction). In some embodiments, UTP is limited during supplementation, e.g., at a concentration such that, upon such supplementation, UTP or a functional analog thereof is present in the reaction at a concentration that is lower than the concentration of one or more (and in some embodiments, all) of ATP or a functional analog thereof, GTP or a functional analog thereof, and/or CTP or a functional analog thereof.

In some embodiments, the functional analog of Nucleoside Triphosphates (NTPs) comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine. In certain embodiments, replacing uridine with modified nucleosides is accomplished by replacing UTP with functional analogs thereof. In some embodiments, the functional analog of UTP is a triphosphate of a modified uridine nucleoside.

In some embodiments, the modified uridine nucleoside is independently selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m 5U).

In some embodiments, the RNA can comprise more than one type of modified uridine nucleoside. In some embodiments, the RNA comprises more than one type of modified uridine nucleoside independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m 1 ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

In some embodiments, the modified nucleoside is any one or more selected from the group consisting of: 3-methyl-uridine (m 3U), 5-methoxy-uridine (mo 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4U), 3-methyluridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-glycolic acid (cmo 5U), uridine 5-glycollic acid methyl ester (mcmo 5U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5U), 5-methoxycarboxymethyl-uridine (mcm 5U), 5-methoxycarboxymethyl-2-thio-uridine (mcm 5s 2U), 5-aminomethyl-2-thio-uridine (nm 5s 2U), 5-methylaminomethyl-uridine (mcm 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mcm 5s 2U), 5-methyl-2-seleno-uridine (mm 5se 2U), 5-carbamoylmethyl-uridine (ncm U), 5-carboxymethyl aminomethyl-uridine (cm 5U), 5-carboxymethyl aminomethyl-2-thio-uridine (cm 5s 2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5s 2U) 1-methyl-4-thio-pseudouridine (m 1s 4. Phi.), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3. Phi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp 3U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp 3. Phi.), 5- (isopentenyl aminomethyl) uridine (mm 5U), 5- (isopentenyl aminomethyl) -2-thio-uridine (mm 5s 2U), alpha-thio-uridine, 2 '-O-methyl-uridine (mm), 5,2' -O-dimethyl-uridine (m 5 Um), 2 '-O-methyl-pseudouridine (psi m), 2-thio-2' -O-methyl-uridine (s 2 Um), 5-methoxy-carboxymethyl-2 '-O-methyl-uridine (mm 5 Um), 5-carbamoyl-methyl-2' -O-uridine (ncm Um), 5-carboxymethyl-2 '-methyl-uridine (mm 5s 2U), alpha-thio-uridine, 2' -O-methyl-uridine (mm), 5 '-O-methyl-uridine (mm), 2' -methyl-uridine (mm), 2 '-O-methyl-pseudouridine (mm), 2' -O-methyl-uridine (mm), 2 '-methyl-uridine (mm-2, mm-methyl-uridine (mm), 2' -O-methyl-uridine (mm-2, mm-uridine (mm), 2-methyl-uridine (mm-uridine, mm-2-methyl-uridine, 3 '-O-methyl-2' -methyl-uridine (mm-3), 2 '-F-uridine, 2' -OH-ara-uridine, 5- (2-carbonylmethoxyvinyl) uridine, 5- [3- (1-E-propenyl amino) uridine or any other modified uridine known in the art.

In some embodiments, the functional analog of NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified adenosine. In certain embodiments, the substitution of the adenosine with the modified nucleoside is accomplished by substituting the ATP with a functional analog thereof. In some embodiments, the functional analog of ATP is a triphosphate of modified adenosine nucleoside.

In some embodiments, the modified nucleoside is any one or more selected from the group consisting of: 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N1-methyl-adenosine, N6-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonyl carbamoyl adenosine, 2-methylsulfanyl-N6-threonyl carbamoyl adenosine, N6-dimethyl adenosine, alpha-thio-adenosine, 8-aza-adenosine, 7-deaza-adenine, 7-methyladenosine, 2-methylsulfanyl-adenine and 2-methoxy-adenine.

In some embodiments, the functional analog of NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified guanosine. In certain embodiments, the substitution of guanosine with a modified nucleoside is accomplished by substituting GTP with a functional analog thereof. In some embodiments, the functional analog of GTP is a triphosphate of modified guanosine nucleoside.

In some embodiments, the modified nucleoside is any one or more selected from the group consisting of: 1-methyl-inosine, hui-guanosine, huai Dinggan, α -thio-guanosine, 6-methyl-guanosine, 7-deazaguanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, O6-methyl-guanosine, N1-methyl-guanosine, N2-dimethyl guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2, N2-dimethyl-6-thio-guanosine.

In some embodiments, the functional analog of NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified cytidine. In certain embodiments, replacing cytidine with a modified nucleoside is accomplished by replacing CTP with a functional analog thereof. In some embodiments, the functional analog of CTP is a triphosphate of a modified cytidine nucleoside.

In some embodiments, the modified nucleoside is any one or more selected from the group consisting of: 5-aza-cytidine, 6-aza-cytidine, α -thio-cytidine, pseudoiso-cytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-hydroxymethyl cytidine, 1-methyl-pseudoiso-cytidine, pyrrolo-pseudoiso-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoiso-cytidine, 4-thio-1-methyl-1-deaza-pseudoiso-cytidine, zebuline, 5-aza-butraline, 5-methyl-zebraline, 5-aza-2-thio-butraline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-thio-1-methyl-pseudocytidine, and 4-methoxy-iso-cytidine.

5' cap

In some embodiments, the RNA produced by the techniques described herein may comprise a cap at its 5' end.

The term "non-extended nucleotide" or "starting nucleotide" or similar terms means a nucleotide that does not have a 5' triphosphate or has a 5' triphosphate that has been modified so that the nucleotide can be incorporated only at the 5' end of the transcript and has a 3' hydroxyl group so that it can be extended at the 3' position. The starting nucleotide is the nucleotide corresponding to the first nucleotide of the RNA. In some embodiments, the addition of the starting nucleotide increases the initiation rate of the RNA polymerase. In certain embodiments, the starting nucleotide is or comprises a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate. In the case where the first nucleotide of the RNA is G, the starting nucleotide may be GTP or GMP or a functional analogue thereof as described herein. In some embodiments, the starting nucleotide is a dinucleotide or a trinucleotide. In some embodiments, the starting nucleotide is nucleoside-5' -triphosphate. In some embodiments, the first nucleotide of the RNA is G, the starting nucleotide is a cap analogue of G, and the corresponding ribonucleoside triphosphate is GTP. In some embodiments, the starting nucleotide is a naturally occurring 5 'cap or 5' cap analog, such as the cap analogs described herein. In some embodiments, the cap is or comprises a guanine nucleotide. These nucleotides may or may not have a cap function. The starting nucleotides include 5 'caps and 5' cap analogs, such as those described herein.

In some embodiments, the RNA produced according to the techniques provided herein (i.e., RNA according to the invention) has an initial nucleotide (starting nucleotide) that is not GTP. In some embodiments, the RNA according to the invention has a starting nucleotide that competes with GTP or a functional analogue thereof for incorporation into the RNA. In some embodiments, the starting nucleotide is as easy to incorporate into the RNA as any other nucleotide. In some embodiments, the starting nucleotide is incorporated into the RNA more efficiently than any other nucleotide, in particular more efficiently than GTP or a functional analogue thereof. In some embodiments, the starting nucleotide is incorporated into the RNA less efficiently than any other nucleotide, in particular less efficiently than GTP or a functional analogue thereof. In some embodiments, the starting nucleotide is supplemented during transcription. In some embodiments, the starting nucleotide is added to the reaction mixture prior to the initiation of the transcription reaction.

In some embodiments, in the reaction mixture used according to the invention, the starting nucleotide corresponding to the first nucleotide of the RNA molecule to be produced is added in excess compared to the fraction of nucleotides predicted to be found or found at the first position of the RNA molecule. In some embodiments, in the reaction mixture, the starting nucleotide corresponding to the first nucleotide of the RNA molecule to be produced is added in excess compared to the fraction of nucleotides competing for incorporation into the RNA molecule. For example, in some embodiments, the first nucleotide is G and the 5 'cap or 5' cap analogue is present in excess relative to GTP in the initial reaction mixture. In some embodiments, the starting nucleotide is added at an initial concentration in the range of about 1 to 20mM, 1 to 17.5mM, 1 to 15mM, 1 to 12.5mM, 1 to 10mM, 1 to 7.5mM, 1 to 5mM, or 1 to 2.5 mM. In some embodiments, the starting nucleotide is added in an excess of at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or even more compared to the nucleotide competing for incorporation into the RNA. For example, in some embodiments, the starting concentration of 5 'cap or 5' cap analogue to the starting concentration of GTP is between about 2:1 and about 20:1, such as about 2:1, 3:1, in some embodiments 4:1, in some embodiments 5:1, in some embodiments 6:1, more in some embodiments 7:1, 8:1, 9:1, 10:1, or even higher.

The terms "5 'cap", "5' cap structure", "cap nucleotide" and "5 'cap nucleotide" are synonymously used to refer to dinucleotides found on the 5' end of some nucleic acids, such as mRNA. A 5' cap is a structure in which the (optionally modified) guanosine is bonded to the first nucleotide of the mRNA molecule via a 5' to 5' triphosphate linkage (or a modified triphosphate linkage in the case of certain cap analogues). In some embodiments, the guanosine is methylated at the 7-position (e.g., a naturally occurring m7G cap). The term "conventional 5 'cap" refers to a naturally occurring RNA 5' cap, and in some embodiments, to a 7-methylguanosine cap (m 7G). Providing an RNA with a 5' cap or 5' cap analogue may be accomplished by in vitro transcription, wherein the 5' cap is co-transcribed into the RNA strand (transcription and capping reaction), or may be attached to the RNA post-transcriptionally (e.g., after in vitro transcription of the RNA) using a capping enzyme, such as a capping enzyme from vaccinia virus or a saccharomyces cerevisiae capping enzyme system. Alternatively, the capped RNA can be obtained by in vitro transcription of a DNA template (IVT), wherein the IVT system contains a 5 'cap or 5' cap analogue in addition to GTP, e.g., as known in the art and described herein. Methods for providing RNAs with 5' caps are well known in the art. In capped RNAs, the 3 'position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base of the RNA molecule ("second base") via a phosphodiester bond.

Those skilled in the art will appreciate that in some embodiments, the addition of a 5' cap to RNA (e.g., mRNA) may facilitate RNA recognition and attachment to ribosomes to initiate translation and enhance translation efficiency. Those skilled in the art will also appreciate that the 5 'cap may also protect the RNA product from 5' exonuclease mediated degradation and thus increase half-life.

As described above, in some embodiments, the RNA produced by the techniques described herein may comprise a cap at its 5' end. In some embodiments, the RNA does not have uncapped 5' -triphosphates. In some embodiments, the RNA can be modified with a 5' cap analog. In some embodiments, the 5' cap is or comprises a synthetic 5' cap analogue that resembles an RNA 5' cap structure and has the ability to stabilize RNA if attached thereto, including for example, but not limited to, anti-reverse cap analogues (ARCA) known in the art and described herein. Those skilled in the art will appreciate that adding a 5' cap to RNA (e.g., mRNA) can facilitate RNA recognition and attachment to ribosomes to initiate translation and enhance translation efficiency. Those skilled in the art will also appreciate that the 5 'cap may also protect the RNA product from 5' exonuclease mediated degradation and thus increase half-life. Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, such as a capping enzyme of a vaccinia virus). In some embodiments, the capped RNA can be obtained by in vitro capping of RNA having a 5 'triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system (including, for example, but not limited to, a vaccinia capping enzyme system or a saccharomyces cerevisiae capping enzyme system). In some embodiments, a capping agent along with a plurality of ribonucleotides may be introduced into an in vitro transcription reaction mixture (e.g., an in vitro transcription reaction mixture as described herein) such that the cap is incorporated into the RNA during transcription (also referred to as co-transcription capping). While it may be desirable in some embodiments to include a 5 'cap in the RNA, in some embodiments the RNA may not have a 5' cap.

The most common method for preparing capped RNA in vitro is to transcribe the DNA template with RNAP such as bacterial or phage RNA polymerase in the presence of all four ribonucleoside triphosphates and a 5 'cap or 5' cap analogue such as m7G (5 ') ppp (5') G (also known as m7 GpppG). RNA polymerase initiates transcription by nucleophilic attack of the 3' -OH of the guanosine portion of m7GpppG on the alpha-phosphate of the next templated nucleoside triphosphate (pppN), resulting in the intermediate m7GpppGpN (where N is the second base of the RNA molecule). The formation of the competitive GTP-initiated product pppGpN is inhibited by adding an excess of 5 'cap or 5' cap analogue relative to GTP, as described herein.

In some embodiments, a 5' capping agent may be added to the in vitro transcription reaction mixture. In some embodiments, the 5' capping agent may comprise a modified nucleotide, such as a modified guanine nucleotide. In some embodiments, the 5 'capping agent may comprise, for example, one or more methyl groups, glyceryl groups, inverted deoxyabasic moieties, 4'5 'methylene nucleotides, L- (beta-D-erythrofuranosyl) nucleotides, 4' thio nucleotides, carbocyclic nucleotides, 1, 5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo- Furanosyl nucleotides, acyclic 3',4' -break nucleotides, acyclic 3, 4-dihydroxybutyl nucleotides, acyclic 3, 5-dihydroxypentyl nucleotides, 3'-3' -inverted nucleotide moieties, 3'-3' -inverted abasic moieties, 3'-2' -inverted nucleotide moieties, 3'-2' -inverted abasic moieties, 1, 4-butanediol phosphate, 3 '-phosphoramidate, hexyl phosphate, 3' -phosphorothioate, phosphorodithioate or bridged or unbridged methylphosphonate moieties, inosine, N1-methyl-guanosine, 2 '-fluoro-guanosine, 7' -deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine. In some embodiments, the 5' capping agent may be or comprise a dinucleotide cap analog (including, for example, m7GpppG cap analog or N7-methyl, 2' -O-methyl-GpppG anti-reverse cap analog (ARCA) cap analog or N7-methyl, 3' -O-methyl-GpppG ARCA cap analog). In some embodiments, the 5' capping agent comprises a 5' N7-methyl-3 ' -O-methylguanosine structure, e.g.Reagent (Trilink BioTechnologies). In some embodiments, the 5 'cap may be or comprise a dinucleotide cap analogue, such as G [5 ]']ppp[5']G、m7G[5']ppp[5']G、m ₃ ^2,2,7 G[5']ppp[5']G、m ₂ ^7,3'-O G[5']ppp[5']G(3'-ARCA)、m ₂ ^7,2'-O GpppG(2'-ARCA)、m ₂ ⁷ ^,2'-O GppSpG (D1) (beta-S-ARCA (D1)) and m ₂ ^7,2'-O GppSpG (D2) (beta-S-ARCA (D2)) and m ₂ ^7,3'-O Gppp (m 2' -O) ApG (CC 413). In some embodiments, the 5 '-capping agent is added in excess to one or more specific ribonucleotides (e.g., GTP, ATP, UTP, CTP or modified versions thereof) to enable incorporation of the 5' -cap into the RNA transcript as a first addition. In some embodiments, the 5' cap used in the present invention is m ₂ ^7,3'-O Gppp(m ^2'-O ) ApG 5' cap.

In the context of the present invention, the term "5 'cap analogue" refers to a molecular structure that is similar to a conventional 5' cap but in some embodiments is modified in vivo and/or in a cell to have the ability to stabilize RNA (if attached thereto). The cap analogue is not a conventional 5' cap.

The 5' cap is generally described as involving efficient translation of mRNA: generally, in eukaryotes, translation is initiated only at the 5' end of the messenger RNA (mRNA) molecule unless an Internal Ribosome Entry Site (IRES) is present. Eukaryotic cells are able to provide RNA with a 5' cap during nuclear transcription: for example, when transcripts reach a length of 20 to 30 nucleotides, newly synthesized mRNA is often modified by a 5' cap structure. First, the 5' terminal nucleotide pppN (ppp means triphosphate; N means any nucleoside) is converted in the cell to 5' GpppN by a capping enzyme having RNA 5' -triphosphatase and guanylate transferase activities. GpppN can then be methylated in the cell by a second enzyme having (guanine-7) -methyltransferase activity to form a monomethylated m ⁷ GpppN cap. In some embodiments, the 5 'cap used in the present invention is a natural 5' cap.

The presence of a cap on an RNA molecule is strongly preferred if it is desired to translate a nucleic acid sequence encoding a protein after introduction of the corresponding RNA into a host cell or host organism, in particular if it is desired to interpret within the first 1 hour or within the first two hours or within the first three hours after introduction of the RNA.

In the present invention, the natural 5' cap dinucleotide is generally selected from the group consisting of: unmethylated cap dinucleotide (G (5 ') ppp (5') N; also referred to as GpppN) and methylated cap dinucleotide ((m) ⁷ G (5 ') ppp (5') N; also known as m ⁷ GpppN)。m ⁷ Gppppn (where N is G) is represented by the formula:

in certain embodiments of the invention, the 5 'cap is a 5' cap analogue. The 5' cap analogues were originally described as facilitating large scale synthesis of RNA transcripts by means of in vitro transcription.

For RNAs such as mRNA, some 5' cap analogs (synthetic caps) have been generally described so far, and they may all be used in the context of the present invention. Desirably, 5' cap analogues are selected that are associated with greater translational efficiency and/or increased resistance to in vivo degradation and/or increased resistance to in vitro degradation.

In some embodiments, 5' cap analogs are used that can only be incorporated into the RNA strand in one orientation. Pasquinelli et al, (1995), RNA J.1:957-967 demonstrated that during in vitro transcription, phage RNA polymerase uses 7-methylguanosine units to initiate transcription, whereby about 40-50% of capped transcripts possess oppositely oriented cap dinucleotides (i.e., when m7G is used, the initial reaction product is Gpppm) ⁷ GpN). Compared to RNA with the correct 5 'cap, RNA with the inverted 5' cap has no function in translating the nucleic acid sequence into a protein. Thus, it is desirable to incorporate the 5' cap in the correct orientation, i.e. to produce a light-emitting device having a cross-section substantially corresponding to m ⁷ RNA of the structure of GpppGpN, etc. Reverse integration of cap-dinucleotides has been shown to be inhibited by substitution of the 2 '-or 3' -OH groups of methylated guanosine units (Stepinski et al, (2001), RNA J.,7:1486-1495; peng et al, (2002), org. Lett., 24:161-164). RNA synthesized in the presence of such "anti-reverse cap analogues" or "ARCA" is compared to that synthesized in the conventional 5' cap m ⁷ RNA transcribed in vitro in the presence of GpppG is translated more efficiently. To this end, for example, holtkamp et al, (2006), blood,108:4009-4017 describe that the 3' OH group of a methylated guanosine unit is OCH-substituted ₃ An alternative cap analogue (7-methyl (3' -O-methyl) GpppG; anti-reverse cap analogue (ARCA)). 7-methyl (3 '-O-methyl) GpppG (sometimes also referred to as 3' -ARCA) is a suitable cap dinucleotide according to the invention.

In some embodiments of the invention, the RNA of the invention is substantially less prone to uncapping. This is important because, in general, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of the mRNA. One in vivo pathway for mRNA degradation begins with removal of mRNA caps. This removal is catalyzed by heterodimeric pyrophosphatase enzymes containing a regulatory subunit (Dcp 1) and a catalytic subunit (Dcp 2). The catalytic subunit cleaves between the alpha and beta phosphate groups of the triphosphate bridge. In the present invention, cap analogs that are insensitive or less sensitive to this type of cleavage may be selected or present. Suitable cap analogues for this purpose may be selected from cap dinucleotides according to formula (I):

Wherein R is ¹ Selected from the group consisting of: optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl,

R ² and R is ³ Independently selected from the group consisting of: H. halo, OH and optionally substituted alkoxy, or R ² And R is ³ Together forming O-X-O, wherein X is selected from the group consisting of: optionally substituted CH ₂ 、CH ₂ CH ₂ 、CH ₂ CH ₂ CH ₂ 、CH ₂ CH(CH ₃ ) And C (CH) ₃ ) ₂ Or R ² And R is R ² The hydrogen atoms at position 4' of the attached ring combine to form-O-CH ₂ -or-CH ₂ -O-，

R ⁵ Selected from the group consisting of: s, se and BH ₃ ，

R ⁴ And R is ⁶ Independently selected from the group consisting of: o, S, se and BH ₃ 。

n is 1, 2 or 3.

R ¹ 、R ² 、R3、R ⁴ 、R ⁵ 、R ⁶ Is disclosed in WO 2011/015347 A1 and may be selected accordingly in the present invention.

For example, in some embodiments of the invention, the 5' cap is or comprises a phosphorothioate-cap-analogue. Phosphorothioate-cap-analogues are those in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom (i.e., R in formula (I) ⁴ 、R ⁵ Or R is ⁶ One of which is S) is a specific cap analogue. Kowalska et al, (2008), RNA,14:1119-1131 have described phosphorothioate-cap-analogs as a solution to the undesirable uncapping process and thus increase the stability of RNA in vivo. In particular, substitution of the oxygen atom for the sulfur atom at the β -phosphate group of the 5' -cap will result in stabilization against Dcp 2. In a preferred embodiment of the invention, R in formula (I) ⁵ S is; and R is ⁴ And R is ⁶ Is O.

In some embodiments of the invention, the RNA of the invention comprises phosphorothioate-cap-analogues in which phosphorothioate modification of the RNA 5' -cap is combined with "anti-reverse cap analogue" (ARCA) modification. Corresponding ARCA-phosphorothioate-cap-analogues are described in WO 2008/157688 A2 and may all be used in the RNAs of the invention. In this embodiment, R in formula (I) ² Or R is ³ At least one of (a) is other than OH, in some embodiments R ² And R is ³ One of them is methoxy (OCH) ₃ ) And in some embodiments, R ² And R is ³ The other of (2) is OH. In some embodiments, an oxygen atom replaces the sulfur atom at the β -phosphate group (such that R in formula (I) ⁵ S is; and R is ⁴ And R is ⁶ Is O). It is believed that phosphorothioate modification of ARCA ensures precise localization of the α, β and γ phosphorothioate groups within the active site of the cap binding protein in both the translation and uncapping mechanisms. At least some of these analogs are substantially resistant to the pyrophosphatase Dcp1/Dcp 2. Phosphorothioate modified ARCAs are described as having a much higher affinity for eIF4E than the corresponding ARCAs lacking phosphorothioate groups.

The corresponding 5' cap analogues (i.e., m ₂ ^7,2'-O Gpp _s pG) is known as β -S-ARCA (WO 2008/157688 A2; kuhn et al, gene Ther., (2010), 17:961-971). Thus, in some embodiments of the invention, the RNA of the invention is modified with beta-S-ARCA (or beta-S-ARCA). beta-S-ARCA is represented by the following structure:

generally, oxygen atoms in place of sulfur atoms at the bridged phosphate will produce phosphorothioate diastereomers designated D1 and D2, based on the elution pattern in HPLC. Briefly, "D1 diastereomer of β -S-ARCA" or "β -S-ARCA (D1)" or "m ₂ ^7,2'-O Gpp _S pG (D1) "is the D2 diastereomer of β -S-ARCA (D2) or m ₂ ^7,2'-O Gpp _S pG (D2)) and therefore exhibit a shorter retention time. The determination of stereochemical configuration by HPLC is described in WO 2011/015347 A1.

In certain embodiments of the invention, the RNA of the invention is modified with the β -S-ARCA (D2) diastereomer. The two diastereomers of β -S-ARCA differ in their sensitivity to nucleases. It has been shown that RNA carrying the D2 diastereomer of β -S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of unmodified ARCA 5 '-cap), whereas RNA with β -S-ARCA (D1) 5' -cap shows intermediate sensitivity to Dcp2 cleavage (71% cleavage). Further, it was shown that increased stability against Dcp2 cleavage correlates with increased protein expression in mammalian cells. In particular, RNAs carrying the β -S-ARCA (D2) cap have been shown to be translated more efficiently in mammalian cells than RNAs carrying the β -S-ARCA (D1) cap. Thus, in some embodiments of the invention, the 5 'cap used in the invention is a 5' cap analogue according to formula (I), characterized by comprising a substituent R in formula (I) ⁵ The stereochemical configuration at the P atom of (2) corresponds to P of the D2 diastereomer of β -S-ARCA _β Stereochemical configuration at the atom. In this embodiment, R in formula (I) ⁵ S is; and R is ⁴ And R is ⁶ Is O. Additionally, in some embodiments, R in formula (I) ² Or R is ³ At least one of which is not OH and/or R ² And R is ³ One of them is methoxy (OCH) ₃ ) And/or R ² And R is ³ The other of (2) is OH; in some embodiments, R in formula (I) ² Or R is ³ Each of (a)None of them is OH; in some such embodiments, R ² And R is ³ One of them is methoxy (OCH) ₃ ) And R is ² And R is ³ The other of (2) is OH.

In certain other embodiments, the 5' cap used in the present invention is the β -S-ARCA (D1) diastereomer. This embodiment is particularly suitable for transferring capped RNA into immature antigen presenting cells, such as for vaccination purposes. beta-S-ARCA (D1) diastereomers have proven particularly suitable for increasing the stability of RNA, increasing the translation efficiency of RNA, extending the translation of RNA, increasing the total protein expression of RNA and/or increasing the immune response against an antigen or antigenic peptide encoded by said RNA when transferring individually capped RNA into immature antigen presenting cells (Kuhn et al, (2010), gene Ther., 17:961-971). Thus, in some embodiments of the invention, the RNA of the invention is modified with a cap analogue according to formula (I) characterized by comprising the substituent R in formula (I) ⁵ The stereochemical configuration at the P atom of (C) corresponds to P of the D1 diastereomer of β -S-ARCA _β Stereochemical configuration at the atom. Corresponding cap analogues and embodiments thereof are described in WO 2011/015347A1 and Kuhn et al, (2010), gene ter, 17:961-971. Any of the cap analogues described in WO 2011/015347A1 (wherein substituent R is comprised ⁵ The stereochemical configuration at the P atom of (C) corresponds to P of the D1 diastereomer of β -S-ARCA _β Stereochemical configuration at the atom) may be used in the present invention. In some embodiments, R in formula (I) ⁵ S is; and R is ⁴ And R is ⁶ Is O. Additionally, in some embodiments, R in formula (I) ² Or R is ³ At least one of which is not OH and/or R ² And R is ³ One of them is methoxy (OCH) ₃ ) And/or R ² And R is ³ The other of (2) is OH; in some embodiments, R in formula (I) ² Or R is ³ Is not OH; in some such embodiments, R ² And R is ³ One of them is methoxy (OCH) ₃ ) And R is ² And R is ³ The other of (2) is OH. In some embodiments, the inventionThe 5' cap used in is m ₂ ^7,3'- ^O Gppp(m ₁ ^2'-O ) ApG (sometimes also referred to as m) ₂ ^7,3`O G(5')ppp(5')m ^2'-O ApG、m ₂ ^7,3'-O Gppp(m ^2'-O ) ApG, CC413, or clearcap). In a particularly preferred embodiment, the RNA of the invention is modified with CleanCap.

5' caps useful in the present invention also include, but are not limited to, m ₃ ^2,2,7 G[5']ppp[5']G、m ₂ ^7,2'-O GpppG(2'-ARCA)、m ⁷ Gp ₃ m ^2'-O G、m ⁷ Gp ₃ m ⁷ G、m2 ^7,2'-O Gp ₃ G、m ₂ ^7,2'-O GpppSG(D1)、m ₂ ^7,2'-O GpppSG(D2)、m ₂ ^7,2'- ^O Gpp _S pG(D1)、m ₂ ^7,2'-O Gpp _S pG(D2)、m ₂ ^7,2'-O GpSppG(D1)、m ₂ ^7,2'-O Gp _S ppG (D2). The 5 'caps useful in the present invention may also be tetraphosphate derivatives of triphosphate 5' cap analogues, such as m ⁷ Gp ₄ G (which is m) ⁷ Gp ₃ Derivatives of G), b ⁷ Gp ₄ G (which is m) ₂ ^7,3'-O Gp ₃ Derivatives of G), b ⁷ m ^3'-O Gp ₄ G (which is b) ⁷ Gp ₃ Derivatives of G), m ₂ ^2,7 Gp ₄ G (which is e ⁷ Gp ₃ Derivatives of G), m3 ^2,2,7 Gp ₄ G (which is m 2) ^2,7 Gp ₃ Derivatives of G), b ⁷ m ² Gp ₄ G (which is m) ₃ ^2,2,7 Gp ₃ Derivatives of G), m ⁷ Gp ₄ m ⁷ G (which is m) ⁷ Gp ₃ Derivatives of 2' dg). Other useful 5' cap analogues have been described in US7074596, WO2008/016473, WO2008/157688, WO 2009/149953, WO2011/015347 and WO 2013/059475.

In some embodiments, the 5 'cap used in the present invention is a 5' cap structure according to formula (I), wherein any one of the phosphate groups is replaced with a borane phosphate group or a selenophosphate group. Such 5' capsHas increased stability in vitro and in vivo. Optionally, the corresponding compounds have a 2 '-O-or 3' -O-alkyl group (wherein alkyl is methyl in some embodiments); the corresponding cap analogue is called BH ₃ -ARCA or Se-ARCA. Capping compounds particularly suitable for mRNA include beta-BH ₃ -ARCA and β -Se-ARCA as described in WO 2009/149953. For these compounds, the substituents R in formula (I) are included ⁵ The stereochemical configuration at the P atom of (C) corresponds to P of the D1 diastereomer of β -S-ARCA _β Stereochemical configuration at the atoms is preferred.

Exemplary in vitro transcription reaction

Those of ordinary skill in the art will understand the materials and reagents used for typical in vitro transcription. In some embodiments, one or more individual reaction components are thawed prior to adding them to the in vitro transcription reaction mixture. For example, an in vitro transcription reaction mixture typically includes a DNA template (e.g., as described herein), ribonucleotides (e.g., as described herein), an RNA polymerase (e.g., a DNA-dependent RNA polymerase), and an appropriate reaction buffer for the selected RNA polymerase. In some embodiments, the in vitro transcription reaction mixture may further comprise an RNase inhibitor. In some embodiments, the in vitro transcription reaction mixture can also comprise pyrophosphatase (e.g., inorganic pyrophosphatase). In some embodiments, the in vitro transcription reaction mixture may further comprise one or more salts (e.g., monovalent and/or divalent salts, such as mg2+), reducing agents (e.g., dithiothreitol, 2-mercaptoethanol, etc.), spermidine, or combinations thereof. In some embodiments, certain reaction components are added in a particular order (e.g., pyrophosphatase and polymerase are added last). In some embodiments, the agitation rate is increased after the addition of a particular reaction component (e.g., pyrophosphatase, polymerase).

Various RNA polymerases suitable for in vitro transcription are known in the art, including, for example, but not limited to, DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or variants or functional domains thereof). The skilled artisan will appreciate that the RNA polymerase used herein may be a recombinant RNA polymerase and/or a purified RNA polymerase, i.e., not as part of a cell extract that contains other components in addition to the RNA polymerase. In some embodiments, the RNA polymerase that can be used for commercial scale in vitro transcription is T7 RNA polymerase. In some embodiments, inorganic pyrophosphatase may be added to increase the yield of in vitro transcription reactions (e.g., in some embodiments, catalyzed by T7 RNA polymerase).

In some embodiments, the buffer used in the transcription reaction (transcription buffer) is optimized for the RNA polymerase of choice. The transcription buffer is typically optimized for the RNA polymerase of choice. For example, in some embodiments, the transcription buffer may comprise Tris-HCl, HEPES, or other suitable buffers. In some embodiments, the transcription buffer may comprise 20-60mM HEPES, 20-60mM divalent salt (e.g., magnesium salt (such as magnesium chloride, magnesium acetate), li ⁺ 、Na ⁺ 、K ⁺ 、NH ⁴⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ Etc.), 5-15mM reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, etc.), and 0.5-3mM spermidine.

In some embodiments, the transcription reaction is performed at a pH of about 6, 6.5, 7, 7.5, 8, or 9. In some embodiments, the transcription buffer has a pH of 7-9 (e.g., about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0). In some embodiments, the transcription buffer has a pH of 6-9. In some embodiments, the pH is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In some embodiments, the transcription buffer has a pH of about 6-8.5. In some embodiments, the buffer has a pH of about 6 to 8.5, about 6.5 to 8.0, about 7.0 to 7.5, in some embodiments about 7.5. In some embodiments, a suitable pH for the transcription reaction may be about 7.5-8.5. In some embodiments, the pH is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In some embodiments, the pH is about 6 to 8.5. In some embodiments, the pH is from 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5; in some embodiments, the pH is 7.5.

In some embodiments, the pH of the reaction mixture is maintained substantially constant during the transcription reaction, for example, by using a suitable buffer. Buffers suitable for adjusting the pH are known in the art and described herein and include, but are not limited to, naOH buffers, KOH buffers, or HCl buffers.

In some embodiments, the pH of the reaction mixture is maintained substantially constant during the transcription reaction, for example, by replenishing a buffer having a pH similar to or equal to the pH of the starting reaction mixture and/or by replenishing a buffer having a pH different from the pH of the starting reaction mixture, if desired.

In some embodiments, the buffer is selected from the group consisting of: 80mM HEPES/KOH pH 7.5 and 40mM Tris/HCl pH 7.5.

Exemplary in vitro transcription reaction conditions

In some embodiments, the in vitro transcription reaction is performed for a period of time, for example, in a bioreactor described herein (selected for a certain in vitro transcription reaction volume, for example, as described herein). In some embodiments, the period of time is at least 20 minutes, including, for example, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 55 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, or at least 180 minutes. In some embodiments, the period of time is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes. In some embodiments, the period of time is about 1.5 to 3 hours. In some embodiments, the period of time is about 25 to 35 minutes.

In some embodiments, the in vitro transcription reaction is performed, for example, in a bioreactor described herein, for a period of time (e.g., as described herein) at a temperature at which the selected RNA polymerase is functionally active. While typical phage RNA polymerases (e.g., T7 polymerase) that perform in vitro transcription reactions are generally inactive at elevated temperatures (e.g., above 45 ℃), thermostable RNA polymerases (e.g., thermostable variants of T7 RNA polymerase, such as those described in US10519431 (the contents of which are incorporated by reference for purposes of this disclosure) can exhibit increased stability at elevated temperatures. In some embodiments, in vitro transcription is performed at a temperature of about 25 ℃ or greater (including, for example, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, or 45 ℃). In some embodiments, in vitro transcription is performed at a temperature of about 45 ℃ or higher (including, for example, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃ or higher).

In some embodiments, in vitro transcription is performed, for example, in a bioreactor described herein, at a pH of about 6, 6.5, 7, 7.5, 8, or 9. In some embodiments, a suitable pH for in vitro transcription may be about 7.5-8.5.

In some embodiments, an in vitro transcription reaction (e.g., in a bioreactor as described herein) performed in accordance with the present disclosure may be performed as a continuous feed reaction; in some embodiments, they may be performed as fed-batch reactions. In some embodiments, one or more nucleotides may be added to the in vitro transcription reaction in a stepwise manner (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more bolus feeds). In some embodiments, the agitation rate is selected such that a specific blending time is achieved to enable rapid mixing of the bolus additives, thereby ensuring optimal availability of the modified nucleotide solution and one or more other nucleotide solutions during RNA synthesis.

Restriction of the reaction components: in certain embodiments, the limiting component is present at a starting concentration that is lower than the starting concentration of the non-limiting component. In some embodiments, the restriction component is a nucleotide, such as ATP, GTP, CTP, UTP or a functional analog thereof. In some embodiments, the non-limiting component is a nucleotide, such as ATP, GTP, CTP, UTP or a functional analog thereof. The limiting or non-limiting component may also be a 5 'cap or 5' cap analogue. In some embodiments, the ratio of limiting component to non-limiting component (such as the ratio of UTP or a functional analog thereof to ATP and/or CTP or a functional analog thereof, or the ratio of GTP or a functional analog thereof to ATP and/or CTP or a functional analog thereof) is between about 1:1 and about 1:100, such as between 1:1.1 and about 1:80, between 1:1.2 and about 1:60, between 1:1.3 and about 1:40, in some embodiments between 1:1.4 and about 1:30, in some embodiments between 1:1.5 and about 1:20, in some embodiments between 1:1.5 and about 1:15, between 1:1.6 and about 1:10, between 1:1.7 and about 1:9, between 1:1.8 and about 1:8, between 1:1.9 and about 1:7, between 1:2 and about 1:6. In some embodiments, the starting concentration of one or more limiting components (such as UTP or a functional analog thereof or GTP or a functional analog thereof) is 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23, 1/24, 1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/31, 1/32, 1/33, 1/34, 1/35, 1/36, 1/37, 1/38, 1/39, 1/40, 1/50, 1/60, 1/70, 1/80, or 1/100 when compared to the starting concentration of the one or more non-limiting components (such as ATP and/or CTP or a functional analog thereof).

UTP restriction and/or supplementation: in some embodiments, the in vitro transcription reaction comprises a limited concentration of UTP or a functional analog thereof in combination with at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof. In some embodiments, the functional analog of UTP is or comprises N1-methyl pseudouridine-5' triphosphate (m 1 ψTP). In some embodiments, UTP or a functional analog thereof is present in the in vitro transcription reaction at an initial concentration that limits the transcription rate. In some embodiments, UTP or a functional analog thereof is present in the in vitro transcription reaction at a starting concentration that is lower than the starting concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof. In some embodiments, the initial concentration of UTP or a functional analog thereof is at least 30% lower (including, e.g., at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower) than the initial concentration of at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof. In some embodiments, the ratio of the starting concentration of UTP or a functional analog thereof to the starting concentration of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally at least one or all of GTP or a functional analog thereof is about 1:1.3 or less, including, for example, 1:1.4, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or less. In some embodiments, the ratio of the starting concentration of UTP or a functional analog thereof to the starting concentration of ATP or a functional analog thereof, CTP or at least one or all of CTP or a functional analog thereof, and optionally GTP or a functional analog thereof is from about 1:1.3 to about 1:20, or from 1:1.5 to about 1:15, or from 1:5 to about 1:15, or from 1:8 to about 1:12. In some such embodiments, the starting concentrations of ATP or a functional analog thereof, CTP or a functional analog thereof, and optionally GTP or a functional analog thereof, may be the same.

In some embodiments, the in vitro transcription reaction is supplemented at least once during the reaction with UTP or a functional analog thereof. In some embodiments, the in vitro transcription reaction is supplemented multiple times (e.g., at least 2 or more times including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times) with UTP or a functional analog thereof during the transcription reaction. In some embodiments, the supplementation is performed when the concentration of UTP or a functional analog thereof in the reaction mixture approaches depletion. In some embodiments, the supplementation is performed when the concentration of UTP or a functional analog thereof in the reaction mixture is less than 100uM, 90uM, 80uM, 70uM, 60uM, 50uM, 40uM, 30uM, 20uM, 10uM, 5uM, 3uM, 2, uM, 1uM, 500nM, 250nM, 200nM, 100nM, 50nM, 25nM or less. In some embodiments, supplementing UTP or a functional analog thereof may include, for example, supplementing UTP or a functional analog thereof and supplementing GTP or a functional analog thereof as a composition optionally comprising additional reactive components as described herein. In other embodiments, replenishing UTP does not refer to replenishing other reaction components. Also, in some embodiments, replenishing GTP does not refer to replenishing other reaction components.

In some embodiments, UTP (or a functional analog thereof) supplementation may be performed continuously during the transcription reaction. For example, in some embodiments, UTP (or functional analogue thereof) replenishment can be performed in a continuous manner at a rate commensurate with (e.g., within 10% or less of) its consumption rate. In some embodiments, UTP (or a functional analog thereof) supplementation may proceed at a rate such that, upon such supplementation, UTP or a functional analog thereof is present in the reaction at a concentration that is lower than the concentration of one or more (and in some embodiments, all) of ATP or a functional analog thereof, GTP or a functional analog thereof, and/or CTP or a functional analog thereof.

In some embodiments, UTP (or a functional analog thereof) supplementation may be performed periodically during the transcription reaction. In some embodiments, UTP (or a functional analogue thereof) supplementation may be performed in a periodic manner such that, after each addition, UTP or a functional analogue thereof is present in the reaction at a concentration that is lower than the concentration of one or more (and in some embodiments, all) of ATP or a functional analogue thereof, GTP or a functional analogue thereof, and/or CTP or a functional analogue thereof. In some embodiments, such periodic replenishment may be performed as one or more boluses or batch additions (including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more boluses or batch additions). In some embodiments, such periodic replenishment may be performed by a fed-batch process. In some embodiments, supplementation may include supplementation of a composition comprising UTP or a functional analog thereof and comprising additional components such as buffer, polymerase, CTP or a functional analog thereof, GTP or a functional analog thereof, ATP or a functional analog thereof, or other components that may be present in a transcription reaction mixture as described herein. In some embodiments, supplementing UTP or a functional analog thereof does not include supplementing CTP or ATP or a functional analog thereof.

In some embodiments, the concentration of UTP or a functional analog thereof added during supplementation is the same as the starting concentration of UTP or a functional analog thereof. In some embodiments, the concentration of UTP or a functional analog thereof added during supplementation is lower than the initial concentration of UTP or a functional analog thereof, e.g., at least 10% lower (including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower) than the initial concentration of UTP or a functional analog thereof.

In some embodiments, the supplementation of UTP (or a functional analogue thereof) is performed at a concentration and/or at a rate or manner such that the ratio of the concentration of UTP or a functional analogue thereof to the concentration of at least one or all of ATP or a functional analogue thereof, CTP or a functional analogue thereof, and optionally GTP or a functional analogue thereof (during the reaction) remains substantially the same (e.g., within 10% or less) as the initial ratio of the concentration of UTP or a functional analogue thereof to the initial concentration of at least one or all of ATP or a functional analogue thereof, CTP or a functional analogue thereof, and optionally GTP or a functional analogue thereof (at the beginning of the reaction).

In some embodiments, UTP or a functional analog thereof is supplemented until the transcription reaction is completed.

In some embodiments, UTP or a functional analog thereof is present in the initial transcription reaction at an initial concentration of 0.1 to 2mM, or 0.1 to 1.5mM, or 0.1 to 1mM, or 0.5 to 2mM, or 1 to 2 mM. In some embodiments, UTP or a functional analog thereof is maintained at a concentration of 0.1 to 2mM, or 0.1 to 1.5mM, or 0.1 to 1mM, or 0.5 to 2mM, or 1 to 2mM during the in vitro transcription reaction.

Optional additional non-UTP restrictions and/or supplements: in some embodiments, at least one of the non-UTPs (or functional analogs thereof) is provided in a limiting concentration (in addition to the limited UTP or functional analog thereof) in an initial in vitro transcription reaction (e.g., at the beginning of an in vitro transcription reaction). For example, in some embodiments, at least one of ATP or a functional analog thereof, CTP or a functional analog thereof, or GTP or a functional analog thereof is provided at a limiting concentration (in addition to the limited UTP or functional analog thereof) in an initial in vitro transcription reaction (e.g., at the beginning of an in vitro transcription reaction). In some embodiments, GTP or a functional analog thereof is provided at a limiting concentration (in addition to the limited UTP or functional analog thereof) in an initial in vitro transcription reaction (e.g., at the beginning of an in vitro transcription reaction).

In certain embodiments of the invention, the method involves supplementing the transcription and capping reactions with GTP or a functional analogue thereof, as it competes with the cap analogue in certain reactions, such as when T7, SP6 or T3 polymerase is used to catalyze the reactions. However, it should be understood that the present invention is not limited to GTP or a functional analogue thereof. In contrast, the invention may be practiced with respect to any reaction involving nucleotides competing with cap analogs or non-extended mononucleotides or dinucleotides that may be incorporated at the 5' end of the transcript. Thus, it is specifically contemplated that any embodiment involving GTP or a functional analog thereof as a competitor nucleotide may be practiced for different nucleotides or nucleotide analogs. The method does not depend on whether GTP and/or a functional analogue thereof is used, as long as the polymerase incorporates the analogue into the prolonged transcript at a rate similar to GTP. The term "functional analogue of GTP" as used herein refers to an extended nucleotide and therefore does not include any cap analogue as defined below.

In some embodiments, the initial concentration of GTP or a functional analog thereof limits the transcription rate. The use of an initial concentration of GTP or a functional analogue thereof that limits the transcription rate of the transcription and/or capping reaction and the supplementation of the reaction with GTP or a functional analogue thereof is preferred, as GTP competes with the 5 'cap or 5' cap analogue in certain reactions, such as reactions using T7, SP6 or T3 polymerase. However, it should be understood that the present invention is not limited to GTP or GTP analogues. In certain embodiments, UTP or a functional analog thereof is present in an amount that limits the onset of the reaction, and the method according to the invention comprises supplementing the reaction mixture with UTP or a functional analog thereof. In certain embodiments, UTP or a functional analog thereof and GTP or a functional analog thereof are present in an amount that limits the onset of the reaction, and the method according to the invention comprises supplementing the reaction mixture with UTP or a functional analog thereof and GTP or a functional analog thereof.

In some embodiments, GTP or a functional analog thereof is present in the in vitro transcription reaction at an initial concentration that limits the transcription rate. In some embodiments, GTP or a functional analog thereof is present in the in vitro transcription reaction at a starting concentration that is lower than the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof. In some embodiments, the starting concentration of GTP or a functional analog thereof is at least 30% lower (including, e.g., at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower) than the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof. In some embodiments, the ratio of the starting concentration of GTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof is about 1:1.3 or less, including, for example, 1:1.4, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 or less. In some embodiments, the ratio of the starting concentration of GTP or a functional analog thereof to the starting concentration of at least one or all of ATP or a functional analog thereof and/or CTP or a functional analog thereof is from about 1:1.3 to about 1:20, or from 1:1.5 to about 1:15, or from 1:5 to about 1:15, or from 1:8 to about 1:12. In some such embodiments, the starting concentration of ATP or a functional analog thereof and/or CTP or a functional analog thereof.

In some embodiments, the in vitro transcription reaction is supplemented at least once during the reaction with GTP or a functional analogue thereof. In some embodiments, the in vitro transcription reaction is supplemented multiple times (e.g., at least 2 or more times including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times) with GTP or a functional analog thereof during the transcription reaction. In some embodiments, the supplementation is performed when the concentration of GTP or a functional analogue thereof in the reaction mixture approaches depletion. In some embodiments, the supplementation is performed when the concentration of GTP or a functional analogue thereof in the reaction mixture is less than 100uM, 90uM, 80uM, 70uM, 60uM, 50uM, 40uM, 30uM, 20uM, 10uM, 5uM, 3uM, 2, uM, 1uM, 500nM, 250nM, 200nM, 100nM, 50nM, 25nM or less.

In some embodiments, GTP (or functional analogue thereof) supplementation may be performed continuously during the transcription reaction. For example, in some embodiments, GTP (or functional analogue thereof) supplementation may be performed in a continuous manner at a rate commensurate with (e.g., within 10% or less of) its consumption rate. In some embodiments, GTP (or functional analogue thereof) supplementation may be performed at a rate such that, upon such supplementation, GTP or functional analogue thereof is present in the reaction at a concentration that is lower than the concentration of ATP or functional analogue thereof and/or CTP or one or more (and in some embodiments, all) of its functional analogues.

In some embodiments, GTP (or functional analogue thereof) supplementation may be performed periodically during the transcription reaction. In some embodiments, GTP (or functional analogue thereof) supplementation may be performed in a periodic manner such that, after each addition, GTP or functional analogue thereof is present in the reaction at a concentration that is lower than the concentration of ATP or functional analogue thereof and/or CTP or one or more (and in some embodiments, all) of its functional analogues. In some embodiments, such periodic replenishment may be performed as one or more boluses or batch additions (including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more boluses or batch additions). In some embodiments, such periodic replenishment may be performed by a fed-batch process. In some embodiments, supplementation may include supplementation of a composition comprising GTP or a functional analogue thereof and comprising additional components (such as buffers, polymerases, CTPs or functional analogues thereof, UTP or functional analogues thereof, ATP or functional analogues thereof, or other components that may be present in a transcription reaction mixture as described herein). In some embodiments, supplementing GTP or a functional analog thereof does not include supplementing ATP or CTP or a functional analog thereof.

In some embodiments, the concentration of GTP or functional analogue thereof added during supplementation is the same as the starting concentration of GTP or functional analogue thereof. In some embodiments, the concentration of GTP or functional analogue thereof added during supplementation is lower than the starting concentration of GTP or functional analogue thereof, e.g. at least 10% (including e.g. at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) lower than the starting concentration of GTP or functional analogue thereof.

In some embodiments, GTP (or functional analogue thereof) supplementation is performed at a concentration and/or at a rate or manner such that the ratio of the concentration of GTP or functional analogue thereof to the concentration of ATP or functional analogue thereof and/or CTP or functional analogue thereof (during the reaction) remains substantially the same (e.g., within 10% or less) as the initial ratio of the concentration of GTP or functional analogue thereof to the initial concentration of ATP or functional analogue thereof and/or CTP or functional analogue thereof (at the beginning of the reaction).

In some embodiments, GTP or a functional analog thereof is supplemented until the transcription reaction is completed.

In some embodiments, GTP or a functional analog thereof is present in the initial transcription reaction at an initial concentration of 0.1 to 2mM, or 0.1 to 1.5mM, or 0.1 to 1mM, or 0.5 to 2mM, or 1 to 2 mM. In some embodiments, GTP or a functional analog thereof is maintained at a concentration of 0.1 to 2mM, or 0.1 to 1.5mM, or 0.1 to 1mM, or 0.5 to 2mM, or 1 to 2mM during the in vitro transcription reaction.

In some embodiments, the non-UTP supplementation does not include supplementation of CTP or a functional analog thereof or ATP or a functional analog thereof.

In some embodiments where non-UTP supplementation is performed, such non-UTP supplementation may be performed simultaneously with UTP supplementation during the reaction. In some embodiments, the non-UTP or a functional analog thereof and UTP or a functional analog thereof may be added to the reaction mixture as a single composition. In some embodiments, the non-UTP or a functional analog thereof and UTP or a functional analog thereof may be added to the reaction mixture as separate compositions, e.g., each at the same or different concentrations and/or each at the same or different flow rates. In some embodiments, such non-UTP supplementation and UTP supplementation may be performed by different methods, e.g., one performed continuously (e.g., as described herein) and the other performed periodically (e.g., as described herein).

In some embodiments, supplementing nucleotides such as UTP and/or GTP or a functional analog thereof includes supplementing more than one type of nucleotide, e.g., supplementing more than one functional analog of UTP and/or GTP. For example, in some embodiments, replenishing the reaction mixture during the transcription reaction includes replenishing UTP and pseudo-UTP. In some embodiments, replenishing the reaction mixture during the transcription reaction includes replenishing UTP, pseudo-UTP, and GTP. In some embodiments, replenishing the reaction mixture during the transcription reaction includes replenishing pseudoutp and/or 1-methyl pseudoutp and GTP. In some embodiments, more than one type of nucleotide is replenished such that a replenishing amount of UTP and/or GTP or a functional analog thereof produces an excess of UTP and/or GTP or a functional analog thereof in the reaction mixture. In some embodiments, supplementing more than one functional analog of UTP and/or GTP does not result in a supplemental amount of UTP and/or GTP or functional analog thereof that produces an excess of UTP and/or GTP or functional analog thereof in the reaction mixture.

In some embodiments, the method for increasing the yield of capped RNA transcripts and/or for reducing dsRNA comprises: the components for the transcription and capping reactions are incubated under conditions that promote the polymerization of the transcripts, wherein the concentration of 5' cap analogue in the reaction is maintained at a ratio of between about 1:1 and about 50:1 relative to the concentration of competing nucleotide component by multiple administrations of competing nucleotide component. In a specific embodiment, the competing nucleotide is GTP or a functional analog thereof. In reactions involving T7, T3 or SP6 RNA polymerase, the competing nucleotide is typically GTP or a functional analogue thereof. It is specifically contemplated that when using an RNA polymerase that employs GTP or a functional analog thereof at the +1 position, any embodiment involving the use of that particular nucleotide may be substituted with another nucleotide triphosphate or a functional analog thereof. The invention also relates to a method for increasing the yield of capped transcripts and/or reducing dsRNA in an in vitro transcription and capping reaction, the method comprising: the reaction components are incubated under conditions capable of transcription, wherein the concentration of GTP or functional analogue thereof in the reaction is maintained at a concentration between about 0.2mM and about 2.0mM, and the concentration of other nucleotides is at least about 0.2mM for at least about 30 minutes during the reaction.

Furthermore, the present invention relates to a method for producing an RNA having a non-extended nucleotide at the 5' end, the method comprising introducing a nucleotide competing with the non-extended nucleotide into a transcription reaction comprising an RNA polymerase and the non-extended nucleotide by a fed-batch process. In certain embodiments, the non-extending nucleotide is not a functional cap analogue. It is specifically contemplated that any of the embodiments discussed with respect to GTP or GTP analogs may be practiced with respect to another nucleotide, so long as the nucleotide competes with the non-extended nucleotide at the 5' end and vice versa. Furthermore, it will also be appreciated that any of the embodiments discussed with respect to the 5' cap or 5' cap analogue may be implemented with respect to non-extended nucleotides that can only be added to the 5' end of the transcript, and vice versa.

In certain embodiments, the reaction may be supplemented with a 5 'cap or 5' cap analogue during transcription and/or capping reactions. In certain embodiments, the reaction is not supplemented with a 5 'cap or 5' cap analogue during the transcription and/or capping reaction.

In some embodiments, for example by fed-batch process to the reaction of the components of one is nucleotide. In some cases, more than one nucleotide is introduced by a fed-batch process. For example, both UTP and GTP nucleotides or functional analogs thereof may be supplemented by a fed-batch process, or UTP and functional analogs thereof may be supplemented by a fed-batch process, and/or GTP and functional analogs thereof may be supplemented by a fed-batch process. In further embodiments, all nucleotides are supplemented by a fed-batch process. The one or more nucleotides in the reaction may be modified nucleotides, such as functional analogues of nucleoside triphosphates described herein. Non-cap nucleotides can be modified but still functional in that they can be incorporated at the 3' end onto the polymerized transcript; that is, these non-cap modified nucleotides are extendible in that they have a 5' triphosphate.

In some embodiments, a programmable pump may be used for replenishment. In some embodiments, programmable syringe pumps may be used, for example, to automatically perform the gradual addition of one or more reaction components. Alternatively or additionally, in some embodiments, a monitor (e.g., a sensor) may be utilized to detect the level of one or more components; in some such embodiments, the monitor may be in automatic communication with the pump, for example, so that additional feed may be released upon detection of a decrease in the amount of such components.

In some embodiments, after RNA transcription, the DNA template may be removed or isolated from the in vitro transcribed RNA composition, for example, using methods known in the art (e.g., DNA hydrolysis).

In some embodiments, an RNase inhibitor may be added during DNA removal or digestion to protect RNA from potential degradation. In some embodiments, a chelating agent may be added to the DNase treated transcription mixture to complex with divalent ions that may be added during an in vitro transcription reaction. An exemplary chelating agent may be or comprise ethylenediamine tetraacetic acid (EDTA). In some embodiments, after addition of the chelating agent, the temperature may be shifted by at least 1 ℃ (including, for example, at least 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃ or more).

Bioreactor

In some embodiments, the transcription reaction is performed in (i.e., using) a bioreactor described herein.

In some embodiments, the transcription reaction is performed at a pH of about 6, 6.5, 7, 7.5, 8, or 9, as described herein. In some embodiments, a suitable pH for the transcription reaction may be about 7.5-8.5. In some embodiments, the pH is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In some embodiments, the pH is about 6 to 8.5. In some embodiments, the pH is from 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5; in some embodiments, the pH is 7.5. In some embodiments, the pH of the reaction mixture is maintained substantially constant during the transcription reaction, for example, by using a suitable buffer. Buffers suitable for adjusting the pH are known in the art and described herein and include, but are not limited to, naOH buffers, KOH buffers, or HCl buffers. In some embodiments, the buffer has a pH as described herein, e.g., 6 to 8.5, 6.5 to 8.0, 7.0 to 7.5, or 7.5. In some embodiments, the buffer is selected from the group consisting of: 80mM HEPES/KOH pH 7.5 and 40mM Tris/HCl pH 7.5. In some embodiments, maintaining the pH constant and/or monitoring the pH during the transcription reaction is accomplished in a bioreactor (i.e., the bioreactor).

In some embodiments, the progress of the transcription reaction is monitored in real time. In some embodiments, monitoring the progress of the transcription reaction is accomplished using a bioreactor (such as a bioreactor comprising a sensor, e.g., a UV flow cell for UV 260/280nm measurement).

The term "bioreactor" or "transcription reactor" as used herein refers to a vessel such as a chamber or tube or column, wherein the transcription reaction is performed under specific conditions such as those described herein. Bioreactors for transcription are known in the art (see WO 1995/08626 and EP 3155 129). The bioreactor is typically configured such that the reaction components are delivered to the reactor core through a feed line, and the RNA product is removed by passing through an ultrafiltration membrane (see EP 3155 129 and van de Merbel, (1999), j. Chromatogrj. A856 (1-2): 55-82) to an outlet stream. The bioreactor useful in the methods of the invention may comprise a reaction module for performing a transcription reaction, a capture module for temporarily capturing transcribed RNA molecules, and a control module for controlling the feeding of components of the reaction mixture into the reaction module, wherein the reaction module may comprise a filtration membrane for separating nucleotides from the reaction mixture, and the controlling of the feeding of components of the reaction mixture by the control module may be based on the measured concentration of the separated nucleotides. The bioreactor may be thermally regulated to accurately maintain a specific temperature, such as the temperature of the transcription reactions described herein, for example, typically between 4 ℃ and 40 ℃. The bioreactor may include an inflow foot and an outlet. The bioreactor may allow for stirring of the reaction mixture during the transcription reaction, for example at a variable stirring rate. Agitation may be continuous or discontinuous, such as intermittent.

The bioreactor used according to the invention may have any size as long as it is available for transcription. For example, in some embodiments, the bioreactor may be at least 0.2 liters or greater, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters or greater, or any volume therebetween. The internal conditions of the bioreactor (including but not limited to pH and temperature) are typically controlled during the transcription reaction as described herein. The bioreactor may be constructed of any material (including glass, plastic or metal) suitable for in vitro transcription under the conditions as described herein.

In some embodiments, the bioreactor may be equipped with a pump for replenishing the reaction mixture. In some embodiments, a programmable pump may be used for replenishment. In some embodiments, programmable syringe pumps may be used, for example, to automatically perform the gradual addition of one or more reaction components. Alternatively or additionally, in some embodiments, a monitor (e.g., a sensor) may be utilized to detect the level of one or more components; in some such embodiments, the monitor may be in automatic communication with the pump, for example, so that additional feed may be released upon detection of a decrease in the amount of such components.

Use of the same

In many embodiments, the RNA products produced by the methods described herein have reduced amounts of dsRNA contaminants as compared to RNA products produced by processes in which UTP is limited in an in vitro transcription process. In some embodiments, such RNA products have low levels of dsRNA contaminants, and no purification process is required to remove the dsRNA contaminants. In some embodiments, such RNA (e.g., mRNA) is therapeutic. In some embodiments, such RNA (e.g., mRNA) is a pharmaceutical grade product.

In some embodiments, one or more RNAs (e.g., single-stranded RNAs) can be formulated with lipid nanoparticles or complexed with liposomes to produce a pharmaceutical grade composition or formulation comprising an RNA-LNP or RNA-lipid complex. In some embodiments, the lipid nanoparticle is a lipid nanoparticle comprising one or more cationic or ionizable lipids, e.g., as known in the art. In some embodiments, the lipid nanoparticle may comprise at least one cationic or ionizable lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid such as a phospholipid and/or sterol).

In some embodiments, such RNA products described herein may be administered to a subject in need thereof (e.g., would benefit from expression of the encoded polypeptide, e.g., as an alternative or to stimulate or enhance an immune response), generally in some embodiments via incorporation into an LNP.

Treatment and vaccination of the condition: in many embodiments, the provided RNA products (e.g., formulations manufactured by using the processes as described herein) are used for treatment of a disorder and/or for vaccination.

The term "immunization" or "vaccination" generally refers to the process of treating a subject for therapeutic or prophylactic reasons. The treatment, particularly the prophylactic treatment, is preferably or includes a treatment intended to induce or enhance an immune response in a subject, for example, to one or more antigens. According to the present invention, if it is desired to induce or enhance an immune response by using an RNA as described herein, the immune response may be triggered or enhanced by the RNA. In some embodiments, the invention provides a prophylactic treatment that preferably is or includes vaccinating a subject. One embodiment of the invention is particularly useful for vaccination, wherein the RNA of the invention encodes a pharmaceutically active peptide or protein, which is an immunologically active compound or antigen, as the protein of interest. When a disorder is intended to be treated, the RNA of the present invention preferably encodes a peptide or protein or nucleic acid (e.g., therapeutic nucleic acid, including but not limited to siRNA, shRNA, miRNA, etc.) that is capable of or sufficient to treat the disorder.

The terms "subject" and "individual" are used interchangeably and refer to a mammal. For example, mammals in the context of the present invention are humans, non-human primates, domestic animals (such as dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (such as mice, rats, rabbits, guinea pigs, etc.), and containment animals (such as zoo animals). The term "animal" as used herein also includes humans. The term "subject" may also include patients, i.e. animals, preferably humans, suffering from a disease.

The agents (e.g., RNA products) and compositions described herein are preferably administered in an effective amount. An "effective amount" refers to an amount that alone or in combination with additional doses achieves a desired response or desired effect. In the case of treating a particular disease or condition, the desired response preferably involves inhibiting the disease process. This includes slowing the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or disorder may also be to delay the onset of the disease or disorder or to prevent the onset of the same.

The effective amount of an agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, body shape and weight), the duration of the treatment, the type of concomitant therapy (if present), the particular route of administration, and the like. Thus, the dosage of agents described herein may depend on several of these parameters. In cases where the response in the patient is insufficient at the initial dose, a higher dose (or effectively a higher dose achieved by a different more localized route of administration) may be used.

Medicine box

The invention provides, inter alia, kits comprising one or more components useful for producing an RNA according to the invention and/or kits comprising an RNA such as produced by the methods of the invention. In some embodiments, the kit may comprise, for example, pharmaceutically acceptable excipients, diluents, carriers, and the like. In some embodiments, the kit comprises a formulation of the RNA product produced as described herein and one or more pharmaceutically acceptable excipients, diluents, carriers, and the like. In some embodiments, kits are provided that include one or more instruments (e.g., syringes or vials or IV bags) or components thereof for administration. In some embodiments, kits are provided that include one or more means for dilution.

In some embodiments, the various components of the kit exist as separate entities. For example, a kit comprising two or more nucleic acid molecules (e.g., two or more RNA products as described herein) can include them in separate containers. Alternatively or additionally, the kit may include one or more nucleic acid molecules in one or more containers, and one or more other components (e.g., buffers, carriers, diluents, excipients, etc.) in one or more containers separate from any container that includes the nucleic acid.

In some embodiments, the individual containers may be open containers or closed containers. In some embodiments, some or all of the containers are closed containers.

In some embodiments, any container comprising RNA or a component to be combined with RNA (e.g., buffer, carrier, diluent, excipient, etc.) is RNAse-free or substantially RNAse-free.

In some embodiments, the kits of the invention comprise RNA for seeding cells and/or for administration to a human or animal subject. In some embodiments, the RNA formulation is frozen. In some embodiments, the RNA formulation is dried. In some embodiments, the RNA formulation comprises a lipid (e.g., LNP).

The kit according to the invention optionally comprises a label or other form of information element, such as an electronic data carrier. The label or information element preferably comprises instructions, for example printed written instructions or optionally instructions in printable electronic form. The instructions may refer to at least one suitable possible use of the kit.

RNA formulations and compositions

As described herein, the present disclosure provides various formulations of RNA products and/or other compositions comprising RNA (e.g., RNA produced as described herein).

Thus, the present disclosure provides compositions obtainable by the methods of the invention, e.g., comparative compositions that contain less double stranded RNA than compositions that are not obtainable by the methods of the invention.

In some embodiments, the provided compositions are purified; in some embodiments, it may not be purified. The composition comprising RNA may also comprise other chemicals and molecules, if not further purified, such as components of the transcription mixture used to transcribe RNA, i.e. DNA template molecules, enzymes, salts, NTPs, etc.

In some embodiments, the composition comprising RNA according to the invention is sufficiently pure for use in subsequent processes without further purification. In some embodiments, a composition comprising RNA according to the invention is sufficiently pure for administration to a cell and/or subject in need thereof. In some embodiments, compositions comprising RNA according to the invention need to be purified after transcription reactions and then used in further processes. In some embodiments, a composition comprising RNA according to the invention is required to be purified after a transcription reaction and then for administration to a cell or subject in need thereof.

In some embodiments, the amount of dsRNA produced by the methods of the invention is reduced compared to the amount of dsRNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analog thereof. In some embodiments, the amount of dsRNA produced by the methods of the invention is reduced by at least 10%, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100%, compared to the amount of dsRNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analogue thereof. Preferably, the amount of dsRNA produced by the methods of the invention is reduced by at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 57%, at least 48%, at least 49%, at least 60%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, preferably at least 57%, at least 58%, at least 59%, at least 60% or more compared to the amount of dsRNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analogue thereof.

In some embodiments, the yield of RNA produced by the methods of the invention is increased compared to the yield of RNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analog thereof. In some embodiments, the yield of RNA produced by the methods of the invention is increased by at least 10%, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or 100%, compared to the yield of RNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analogue thereof. Preferably, the yield of RNA produced by the method of the invention is increased by at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 57%, at least 48%, at least 49%, at least 60%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, preferably at least 57%, at least 58%, at least 59%, at least 60% or more compared to the yield of RNA produced by a method using an equimolar amount of ATP, CTP, GTP, UTP or a functional analogue thereof.

Pharmaceutical composition

In some embodiments, the RNA of the invention (such as the RNA produced by the methods of the invention) may be present in the form of a pharmaceutical composition. The pharmaceutical composition according to the invention may comprise at least one RNA molecule according to the invention. The pharmaceutical compositions according to the present invention may further comprise any one or more of pharmaceutically acceptable diluents, excipients, carriers and/or vehicles. The choice of pharmaceutically acceptable carrier, vehicle, excipient or diluent is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient or diluent known in the art may be used.

The term "carrier" refers to an organic or inorganic component of natural or non-natural (synthetic) nature with which the active component is combined in order to facilitate, enhance or effect application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.

Possible carrier substances for parenteral administration are, for example, sterile water, dextrose solution, ringer's solution, lactated ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, in particular biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The pharmaceutical compositions described herein may be administered via any conventional route, such as by parenteral administration, including by injection or infusion. Administration is preferably parenteral, e.g., intravenous, intra-arterial, subcutaneous, intra-lymph node, intradermal, or intramuscular.

In some embodiments of the invention, the pharmaceutical composition may further comprise a solvent, such as an aqueous solvent or any solvent that can maintain RNA integrity. In some embodiments, the pharmaceutical composition is an aqueous solution comprising RNA. The aqueous solution may optionally comprise a solute, such as a salt.

In some embodiments of the invention, the pharmaceutical composition is in the form of a lyophilized composition. The lyophilized composition may be obtained by lyophilizing the corresponding aqueous composition.

Compositions suitable for parenteral administration typically comprise a sterile aqueous or non-aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspension medium.

In some embodiments, the pharmaceutical composition comprises at least one cationic entity. Generally, cationic lipids, cationic polymers, and other positively charged species can form complexes with negatively charged nucleic acids. The RNA according to the invention can be stabilized by complexation with a cationic compound, preferably a polycationic compound, such as a cationic or polycationic peptide or protein. In some embodiments, the pharmaceutical composition according to the invention comprises at least one cationic molecule selected from the group consisting of: protamine, polyethylenimine, poly-L-lysine, poly-L-arginine, histone or cationic lipids.

In some embodiments, the cationic lipids useful according to the present invention are cationic amphiphilic molecules, such as molecules comprising at least one hydrophilic and lipophilic moiety. In some embodiments, the cationic lipid may be mono-cationic or polycationic. Cationic lipids generally have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and have an overall net positive charge. The head group of a lipid typically carries a positive charge. The cationic lipid preferably has a positive charge of 1 to 10, more preferably 1 to 3, and more preferably 1. Examples of cationic lipids include, but are not limited to, 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA); dimethyl Dioctadecyl Ammonium (DDAB); 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP); 1, 2-diacyloxy-3-dimethylammonium propane; 1, 2-dialkoxy-3-dimethylammonium propane; dioctadecyl dimethyl ammonium chloride (DODAC), 1, 2-dimyristoyloxy propyl-1, 3-dimethylhydroxyethyl ammonium (DMRIE), and 2, 3-dioleyloxy-N- [2 (spermidine) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). Cationic lipids also include lipids having tertiary amine groups, including 1, 2-diiodoyloxy-N, N-dimethyl-3-aminopropane (DLinDMA). Cationic lipids are suitable for formulating RNA in lipid formulations as described herein (such as liposomes, emulsions, and lipid complexes). Typically, positive charges are provided by at least one cationic lipid, while negative charges are provided by RNA. In some embodiments, the pharmaceutical composition comprises at least one helper lipid in addition to the cationic lipid. The helper lipid may be a neutral or anionic lipid. The helper lipid may be a natural lipid (such as a phospholipid) or an analogue of a natural lipid or a fully synthetic lipid or a lipid-like molecule that has no similarity to a natural lipid. In the case where the pharmaceutical composition includes both the cationic lipid and the auxiliary lipid, the molar ratio of the cationic lipid to the neutral lipid may be appropriately determined in view of the stability of the formulation and the like.

In some embodiments, the pharmaceutical composition according to the invention comprises protamine. In some embodiments, protamine may be used as a cationic carrier agent. The term "protamine" refers to any of a variety of strongly basic proteins that are relatively low molecular weight rich in arginine and that have been found to be particularly relevant to DNA to replace the somatic histones in animal (such as fish) sperm cells. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly alkaline, soluble in water, non-coagulating by heat, and comprises multiple arginine monomers. According to the present invention, the term "protamine" as used herein is meant to encompass any protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and multimeric forms of said amino acid sequence or fragments thereof. Furthermore, the term encompasses (synthetic) polypeptides, which are artificial and specifically designed for a specific purpose and cannot be isolated from natural or biological sources.

In some embodiments, the compositions provided herein may comprise one or more adjuvants. Adjuvants may be added to the vaccine to stimulate the immune system's response; adjuvants themselves generally do not provide immunity. Exemplary adjuvants include, but are not limited to, the following: inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide); mineral oil (e.g., paraffin oil), cytokines (e.g., IL-1, IL-2, IL-12); immunostimulatory polynucleotides (such as RNA or DNA; e.g., cpG-containing oligonucleotides); saponins (e.g., plant saponins from quillaja, soybean, senegal); oil emulsions or liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene (PCPP); muramyl peptide; an imidazoquinolone compound; thiosemicarbazone compounds; flt3 ligand (WO 2010/066418 A1); or any other adjuvant known to those skilled in the art. A preferred adjuvant for administration of RNA according to the invention is Flt3 ligand (WO 2010/066418 A1). When Flt3 ligand is administered with antigen-encoding RNA, a strong increase in antigen-specific CD8+ T cells can be observed.

In some embodiments, the pharmaceutical compositions provided herein can be buffered (e.g., with acetate buffer, citrate buffer, succinate buffer, tris buffer, phosphate buffer).

Host cells

In some embodiments, the pharmaceutical (or other) composition is formulated appropriately for introduction into the cell; the cell into which one or more RNA molecules can be inoculated (i.e., administered) can be referred to as a "host cell". As used herein, the term "host cell" refers to any cell that can be transformed or transfected with an exogenous RNA molecule. In many embodiments, the term "cell" is an intact cell, i.e., a cell with an intact membrane that does not release its normal intracellular components such as enzymes, organelles, or genetic material. In many embodiments, the intact cells are living cells, i.e., living cells capable of performing their normal metabolic functions. According to the present invention, the term "host cell" includes prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells). Exemplary cells include prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of E.coli, bacillus, streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae, schizosaccharomyces pombe, pichia pastoris, pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus infected insect cells, spodoptera frugiperda, etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadromas). In some embodiments, the host cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the host cell is a eukaryotic cell. For example, the eukaryotic host cell may be CHO (e.g., CHO Kl, DXB-1 CHO, veggie-CHO), COS (e.g., COS-7), retinal cells, vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, haK, BHK), heLa, hepG2, WI38, MRC 5, colo205, HB 8065, HL-60 (e.g., BHK 21), jurkat, daudi, A (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived therefrom. Of particular interest are mammalian cells, such as cells from humans, mice, hamsters, pigs, domesticated animals (including horses, cattle, sheep, and goats), and primates. Cells may be derived from a variety of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In some embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, monocyte, or macrophage. In some embodiments, the compositions (e.g., pharmaceutical compositions) provided herein can deliver a nucleic acid (e.g., RNA) to a host cell such that it is present in the host cell in a single or several copies, and in some embodiments expressed in the host cell.

In some embodiments, the host cell may be a prokaryotic cell; in some embodiments, the host cell may be a eukaryotic cell.

In some embodiments, prokaryotic cells are used herein for, e.g., proliferation of DNA according to the invention, and eukaryotic cells are suitable herein for, e.g., expression of open reading frames of replicons.

Certain embodiments

The following describes certain embodiments provided by the present disclosure:

1. a method of producing RNA, the method comprising transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein an initial concentration of UTP or a functional analog thereof is lower than an initial concentration of CTP and/or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with a composition comprising UTP or a functional analog thereof and substantially free of CTP or ATP or a functional analog thereof during the transcription reaction.

2. A method of producing RNA, the method comprising transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of CTP or a functional analog thereof is equal to the initial concentration of ATP or a functional analog thereof, and wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with UTP or a functional analog thereof during the transcription reaction.

3. A method of producing a composition comprising RNA having a reduced double stranded (ds) RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP and/or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with a composition comprising UTP or a functional analog thereof and substantially no CTP or ATP or functional analog thereof during the transcription reaction.

4. A method of producing a composition comprising RNA having a reduced double stranded (ds) RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mixture comprising Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or a functional analog thereof, wherein the initial concentration of CTP or a functional analog thereof is equal to the initial concentration of ATP or a functional analog thereof, and wherein the initial concentration of UTP or a functional analog thereof is lower than the initial concentration of CTP or ATP or a functional analog thereof, wherein the method comprises supplementing the reaction mixture with UTP or a functional analog thereof during the transcription reaction.

5. The method of embodiment 3 or 4, wherein the double stranded (ds) RNA content of the RNA-containing composition is reduced compared to the dsRNA content of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

6. The method of any one of embodiments 3-5, wherein the immunogenicity of the composition comprising RNA is reduced compared to the immunogenicity of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

7. The method of any one of embodiments 1 to 6, wherein Uridine Triphosphate (UTP) or a functional analog thereof is present at an initial concentration that limits transcription rate.

8. The method of any one of embodiments 1 to 7, wherein the ratio of the starting concentration of Uridine Triphosphate (UTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

9. The method of any one of embodiments 1 to 8, wherein the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analog thereof when the concentration of UTP or a functional analog thereof is near depletion.

10. The method of any one of embodiments 1 to 9, wherein the reaction mixture is supplemented at least once with Uridine Triphosphate (UTP) or a functional analogue thereof during the transcription reaction.

11. The method of any one of embodiments 1 to 10, wherein the reaction mixture is continuously replenished with Uridine Triphosphate (UTP) or a functional analogue thereof during the transcription reaction.

12. The method of any one of embodiments 1 to 10, wherein the reaction mixture is periodically replenished with Uridine Triphosphate (UTP) or a functional analogue thereof during the transcription reaction.

13. The method of any one of embodiments 1 to 12, wherein the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analog thereof to maintain or restore an initial ratio of concentration of UTP or a functional analog thereof to concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

14. The method of any one of embodiments 1 to 13, wherein the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analogue thereof, until the transcription reaction is completed.

15. The method of any one of embodiments 1 to 14, wherein the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof is lower than the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

16. The method of embodiment 15, wherein Guanosine Triphosphate (GTP) or a functional analog thereof is present at an initial concentration that limits the transcription rate.

17. The method of embodiment 15 or 16, wherein the ratio of the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

18. The method of any one of embodiments 15 to 17, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

19. The method of embodiment 18, wherein the reaction mixture is replenished with Guanosine Triphosphate (GTP) or a functional analog thereof when the concentration of GTP or a functional analog thereof approaches depletion.

20. The method of any one of embodiments 15 to 19, wherein the reaction mixture is supplemented at least once with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

21. The method of any one of embodiments 15 to 20, wherein the reaction mixture is continuously replenished with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

22. The method of any one of embodiments 15 to 20, wherein the reaction mixture is periodically replenished during the transcription reaction with Guanosine Triphosphate (GTP) or a functional analog thereof.

23. The method of any one of embodiments 15 to 22, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analog thereof to maintain or restore an initial ratio of the concentration of GTP or a functional analog thereof to the concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

24. The method of any one of embodiments 15 to 23, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof until the transcription reaction is over.

25. The method of any one of embodiments 1-24, wherein said method does not comprise supplementing said transcription mixture with Cytidine Triphosphate (CTP) and/or Adenosine Triphosphate (ATP) or a functional analog thereof during said transcription reaction.

26. The method of any one of embodiments 1 to 25, wherein the reaction mixture comprises a starting nucleotide corresponding to a first nucleotide in the RNA molecule.

27. The method of embodiment 26, wherein the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

28. The method of embodiment 26 or 27, wherein the starting nucleotide is a 5 'cap or 5' cap analog.

29. The method of embodiment 28, wherein the 5' cap analog is selected from the group consisting of: g < 5']ppp[5']G、m7G[5']ppp[5']G、m ₃ ^2,2,7 G[5']ppp[5']G、m ₂ ^7,3'-O G[5']ppp[5']G(3'-ARCA)、m ₂ ^7,2'-O GpppG(2'-ARCA)、m ₂ ^7,2'-O Gpp _S pG D1(β-S-ARCA D1)、m ₂ ^7,2'-O Gpp _S pG D2 (. Beta. -S-ARCA D2) and m ₂ ^7,3'-O Gppp(m ^2'-O )ApG(CC413)。

30. The method of embodiment 28 or 29 wherein said 5 'cap or 5' cap analogue in said reaction mixture is present in excess compared to Guanosine Triphosphate (GTP) or a functional analogue thereof.

31. The method of embodiment 30, wherein the ratio of the starting concentration of the 5 'cap or 5' cap analogue to the starting concentration of Guanosine Triphosphate (GTP) or a functional analogue thereof is between about 2:1 and about 20:1.

32. The method of embodiment 31, wherein the ratio of the starting concentration of the 5 'cap or 5' cap analog to the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof is about 4:1.

33. The method of any one of embodiments 1 to 32, wherein the reaction mixture further comprises an RNA polymerase, a buffer, and at least one monovalent or divalent cation.

34. The method of embodiment 33, wherein the cation is Li ⁺ 、Na ⁺ 、K ⁺ 、NH ⁴⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ 。

35. The method of embodiment 33 or 34, wherein the RNA polymerase is selected from the group consisting of: t7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.

36. The method of any one of embodiments 1 to 35, wherein the functional analog of Uridine Triphosphate (UTP) is selected from the group consisting of: pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP and 4-thio-UTP.

37. The method of any one of embodiments 1 to 36, wherein the functional analog of Guanosine Triphosphate (GTP) is selected from the group consisting of: 7-deaza-GTP, N1-methyl-GTP and O6-methyl-GTP.

38. The method of any one of embodiments 1 to 37, wherein the DNA template encodes one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

39. The method of any one of embodiments 1 to 38, wherein the RNA comprises one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

40. The method of embodiment 39, wherein the RNA encodes at least one peptide or protein.

41. The method of any one of embodiments 1 to 40, wherein the RNA is mRNA.

42. The method of any one of embodiments 1 to 41, wherein the pH of the reaction mixture is maintained substantially constant during the transcription reaction.

43. The method of any one of embodiments 1 to 42, wherein the progress of the transcription reaction is monitored in real time.

44. The method of any one of embodiments 1 to 43, wherein the method is performed using a bioreactor.

45. An RNA produced by the method of any one of embodiments 1 to 44.

46. A composition comprising RNA produced by the method of any one of embodiments 3 to 44.

47. A method of treating a subject, the method comprising the steps of:

(i) Obtaining RNA produced by the method of any one of embodiments 1 to 44, or obtaining a composition comprising RNA produced by the method of any one of embodiments 3 to 44, and

(ii) Administering the RNA or the composition comprising RNA to the subject.

48. A method of treating a subject by administering to the subject the RNA of embodiment 45 or a composition comprising the RNA of embodiment 46.

49. In a method for producing RNA by in vitro transcription, the improvement comprising:

limiting the concentration of UTP or a functional analogue thereof during said in vitro transcription reaction.

50. An in vitro transcription reaction comprising:

limiting the concentration of UTP or a functional analogue thereof during said in vitro transcription reaction. An in vitro transcription reaction comprising:

An RNA template comprising a promoter that directs transcription of the template to produce a transcript having a polyA sequence element;

an RNA polymerase acting on the promoter; and

adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP) and Uridine Triphosphate (UTP) or functional analogues thereof, wherein the initial concentration of UTP or functional analogue thereof is lower than the concentration of CTP and/or ATP or functional analogue thereof.

Illustration of an example

The invention is described and illustrated in detail by the figures and examples, which are for illustrative purposes only and are not meant to be limiting. Other embodiments that are also encompassed by the present invention will be available to those of skill in the art from the description and examples.

Example 1: exemplary IVT reactions that limit UTP or UTP and ATP in transcription reactions can reduce dsRNA content Is generated by (a)

This example demonstrates an exemplary fed-batch procedure to enhance capping efficiency and/or manipulate the amount of double stranded RNA (dsRNA) content generated during an In Vitro Transcription (IVT) reaction. In some embodiments, the dsRNA is produced by reverse transcription (e.g., 3 'to 5' direction). In some embodiments, limiting the NTP required to initiate transcription from the 3' end minimizes this effect. This example demonstrates in particular that limiting the UTP in vitro transcription reaction can reduce dsRNA formation and can be particularly useful for producing transcripts that can include polyA sequences (such as polyA tails). Without wishing to be bound by theory, we propose that the observed reduction in dsRNA production may be due to a reduction in reverse transcription (e.g., initiation upon hybridization to a polyA sequence (such as a polyA tail)).

To test the effect of limiting NTP during IVT, ATP (a), UTP (U) and a combination of ATP and UTP (a/U) were fed into the reaction at intervals. Standard GTP (G) feed was used as a control.

In some embodiments, the starting concentration of G, U, A or a/U is reduced to 20% of its respective starting concentration (e.g., typical starting concentration used in IVT), and the feed is added in 4 portions during the transcription reaction until the final concentration is reached.

Exemplary IVT in DNA template, m for Co-transcriptional capping ₂ ^7,3'-O Gppp(m ^2'-O ) ApG (CC 413) cap analogs and nucleoside triphosphates (GTP, ATP, UTP, CTP) are present. The IVT reaction was performed in the presence of T7 RNA polymerase, RNAse inhibitor (Ribolock) and inorganic pyrophosphatase for 150 minutes with magnesium acetate buffer containing dithiothreitol and spermidine.

In some embodiments, after IVT, the DNA template is removed (e.g., via DNAse digestion) and the RNA is purified (e.g., using magnetic beads for immobilization (Berensmeier, S. Magnetic particles for the separation and purification of nucleic acids. Appl. Microbiol. Biotechnol.73,495-504;10.1007/s00253-006-0675-0 (2006)), in some embodiments, the RNA is eluted, e.g., in H2O).

In addition to assessing dsRNA content, yield, RNA integrity and capping efficiency of IVT were also determined to characterize RNAs derived from different transcription conditions.

In some embodiments, the amount of dsRNA is determined. In some embodiments, 1 μg of RNA is spotted onto a nylon blotting membrane (Nytran SuPerCharge (SPC) nylon blotting membrane) in 5 μl aliquots. The membrane is then blocked in buffer (e.g., TBST-20mM TRIS pH 7.4, 137mM NaCl, 0.1% (v/v) TWEEN-20) containing skimmed milk powder (e.g., 5% (w/v)) for 1 hour. For detection of dsRNA, the membrane is incubated with dsRNA-specific antibodies (e.g., mouse monoclonal antibodies) diluted 1:10,000 in buffer (e.g., TBS-T buffer containing 1% (w/v) skimmed milk powder) for 1 hour. After washing with buffer, the membrane was incubated with a secondary antibody (e.g., HRP conjugated donkey anti-mouse IgG) diluted 1:10,000 in buffer (e.g., TBS-T containing 1% (w/v) skimmed milk powder) for 1 hour, washed with buffer (e.g., TBS-T), and developed using Western blot detection reagents and imaging systems. In some embodiments, hybridization signal intensity is quantified, if indicated, for example, by densitometry.

In some embodiments, RNA concentration is measured, for example, by UV (e.g., nanodrop) and IVT yield is calculated (e.g., produced RNA in μg/IVT reaction volume in μl).

In some embodiments, the RNA integrity is analyzed using a Bioanalyzer (e.g., agilent). To prepare an exemplary sample for RNA integrity analysis, 250ng RNA in 50% formamide was denatured at 70 ℃ for 10 minutes and further treated with Agilent RNA 6000Nano Kit (5067-1511, agilent). In some embodiments, the integrity is calculated later by the relationship of the main peak integral to the integral of the complete electropherogram.

In some embodiments, RNA capping efficiency is determined. In some embodiments, RNA is treated with RNA ribozymes to cleave RNA in the 5' utr, and fragments are purified on denaturing gels (e.g., 21% polyacrylamide), resolving the 1nt difference between capped and uncapped fragments. In some embodiments, the ratio between capped and uncapped fragments is then determined by optical density methods.

In some embodiments, the feeding of different nucleotides (G, A, U or a/U) has a slight/negligible effect on RNA integrity and RNA yield (fig. 1A and B).

In some embodiments, a dramatic decrease in dsRNA content compared to the control (GTP feed) is observed when UTP is limited (fig. 1C). In addition, the opposite effect (e.g., increase) is observed when ATP is limited (fig. 1C). Without wishing to be bound by any one theory, the increase in dsRNA content observed when limiting ATP may be due to T7 polymerase stalling at the ends of the transcript, as there may not be enough ATP available to synthesize the polyA tail at typical rates (e.g., where ATP is not limited). This stagnation may be advantageous for reverse transcription due to the longer residence time of the polymerase at the transcript ends. The increase in dsRNA content observed when ATP was limited was rescued by feeding ATP and UTP simultaneously (e.g., dsRNA content was reduced to control levels) (fig. 1C).

In some embodiments, the limitation of GTP is used to increase capping efficiency. In some embodiments, the limitations of other NTPs may have different effects on the ratio of capped RNA oligonucleotides. Although the exemplary capping analog CC413 was designed for high capping efficiency, the confinement of GTP showed the highest capping efficiency compared to the confinement of other NTPs (fig. 1D). In some embodiments, the limitation of UTP or ATP results in reduced capping efficiency compared to the control. In some embodiments, the dual limitation of ATP and UTP only shows a slight decrease in capping efficiency compared to the control (fig. 1D).

Example 2: exemplary fed-batch additions of UTP and/or GTP may save capping efficiency

This example demonstrates that capping efficiency can be restored by limiting the initial concentration of UTP or by limiting the initial concentration of both UTP and GTP (G/U), while reducing the production of dsRNA content.

In some embodiments, incorporating a cap analog at the 5' end of the RNA competes with incorporating GTP. In some embodiments, capping is most efficient when Cap analogs are in excess relative to GTP in an IVT reaction. In some embodiments, this may be achieved by maintaining a low GTP concentration throughout the IVT reaction, but may reduce yield and RNA integrity. Without being bound by any one theory, the reduced yield and RNA integrity may be due to low GTP concentration, which limits the reaction rate and leads to transcription failure. In some embodiments, yield and RNA integrity is improved by stepwise addition of GTP to the reaction to keep total GTP concentration low while always providing enough GTP to maintain transcription efficiency. In some embodiments, this is even more important for the use of non-trinucleotide cap analogues (such as ARCA or β -SARCA-D1 cap analogues). For this example, β -SARCA-D1 was used. To save capping efficiency in the UTP feed reaction, GTP is fed in addition to UTP. Standard GTP feed was used as a control.

For example, the starting concentrations of GTP, UTP and GTP/UTP are reduced to 1/18 of the starting concentration and the feed is added 17 times during the transcription reaction until the final concentration is reached.

Exemplary IVT is performed in the presence of a DNA template, a beta-S ARCA (D1) cap analogue for co-transcription capping, and nucleoside triphosphates (GTP, ATP, UTP, CTP).

Exemplary reactions were performed for 180 minutes with magnesium acetate buffer containing dithiothreitol and spermidine in the presence of T7 RNA polymerase, RNAse inhibitor (Ribolock) and inorganic pyrophosphatase. RNA was purified and assayed for RNA concentration, integrity, dsRNA content, and capping efficiency as described in example 1.

In some embodiments, the limitation of UTP and the dual limitation of GTP and UTP results in increased yield compared to the control (fig. 2A). In some embodiments, the integrity is reduced by limiting UTP, but when GTP and UTP are limited together, the integrity is restored to the control level (fig. 2B). It was similarly observed in example 1 that dsRNA content was reduced during IVT by limiting UTP, as compared to limiting GTP alone or both GTP and UTP together. Dual GTP and UTP restrictions allow for reduced dsRNA content, but to a lesser extent than UTP-only conditions (FIG. 2C). In some embodiments, the goal of the dual restriction is to save capping efficiency. While the limitation of UTP reduced capping efficiency, the dual limitation of GTP and UTP restored capping efficiency to a level comparable to the control reaction (fig. 2D).

Example 3: exemplary fed-batch additions of m1ψTP and/or GTP can save capping efficiency

This example demonstrates that limiting the starting concentration of 1-methyl-pseudouridine (m 1 ψtp) or both m1 ψtp and GTP (G/ψ) can reduce dsRNA production during IVT compared to the starting concentration of GTP only (control), whereas limiting the starting concentration of both m1 ψtp and GTP can maintain dsRNA reduction and maintain capping efficiency.

In some embodiments, the use of m1 ψtp instead of UTP reduces the immunogenicity of in vitro transcribed RNA and the amount of dsRNA generated in the IVT reaction. To determine if dsRNA levels can be further reduced, m1ψtp was limited as with UTP in examples 1 and 2. Standard GTP feed was used as a control.

In some embodiments, the starting concentrations of GTP, m1ψtp, and GTP/m1ψtp are reduced to 1/11 of the starting concentration and the feed is added in 10 portions during the transcription reaction until the final concentration is reached.

Exemplary IVT uses T7 polymerase in DNA template, m for Co-transcriptional capping ₂ ^7,3'-O Gppp(m ^2'-O ) ApG (CC 413) cap analogs and nucleoside triphosphates (GTP, ATP, m 1.1.psi. TP, CTP) are performed in the presence of a primer.

Exemplary reactions were performed for 180 minutes with magnesium acetate buffer containing dithiothreitol and spermidine in the presence of T7 RNA polymerase, RNAse inhibitor (Ribolock) and inorganic pyrophosphatase.

RNA was purified as described in example 1 and assayed for RNA yield, integrity and dsRNA content. Capping efficiency was determined by digesting RNA with 3' exonuclease and measuring the remaining nucleotides by mass spectrometry. Capping efficiency was calculated by the ratio of ATP and GTP to cap analogue.

In some embodiments, the feeding of different nucleotides has only a modest effect on RNA yield (fig. 3A). In some embodiments, RNA integrity is reduced by limiting m1ψtp, but when GTP and m1ψtp are limited together, the integrity is unchanged from control (fig. 3B). Similar to that observed in example 1, in some embodiments dsRNA contamination was reduced by limiting the starting concentration of m1 ψtp (UTP in example 1). In some embodiments, limiting the starting concentration of both GTP and m1 ψtp maintains a similar reduction in dsRNA contamination generated compared to that observed under conditions limiting only m1 ψtp (fig. 3C). Similar to that observed in the UTP fed-batch procedure (example 1), capping efficiency was reduced compared to the control when m1 ψtp was limited, but the double limitation by both m1 ψtp and GTP was restored to the control level (fig. 3D).

Example 4: exemplary body Scheme for preparation of externally transcribed RNA

Initial in vitro transcription reaction: the components were combined in a reaction vessel under stirring, including ATP solution (100 mM adenosine 5 '-triphosphate), CTP solution (100 mM cytidine 5' -triphosphate), N1-methylpseudoup solution (100 mM N1-methylpseudoup 5 '-triphosphate), GTP solution (100 mM guanosine 5' -triphosphate), 5 '-cap solution (100 mM 5' -cap), RNase inhibitor, transcription buffer (10X 400mM HEPES, 400mM magnesium acetate, 100mM DTT, 20mM spermidine, pH 8.3) and linear DNA template (water for injection (WFI)).

Pyrophosphatase and T7 polymerase (T7 RNA polymerase) were then added and agitation was increased. The total volume of the initial reaction is typically about 30L, for example more than 35L, for example between about 30L and about 50L or between about 35L and about 45L.

After enzyme addition, an incubation period begins during which there is a GTP/N1-methyl pseudo UTP bolus feed.

Primary incubation and fed-batch: during the incubation period, an equal amount of a mixture of N1-methyl pseudo UTP and GTP was delivered as a bolus feed. Multiple bolus feeds may be added during the incubation period. For example, the feed may be added at an average rate of about 1 time, for example, every 4-7 minutes or every 5-10 minutes, or as desired. In some embodiments, the first incubation period may last more than about 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, and the like. In some embodiments, the first incubation period is from about 60 to about 80 minutes, or from about 65 to about 75 minutes, or from about 65 to about 70 minutes. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more feeds are performed during the first incubation period.

The total volume after all additional feeds will be increased (relative to the volume of the initial reaction) to, for example, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 or more. In some embodiments, the total volume will increase from about 2 to about 8 times, or from about 3 to about 7 times, or from about 4 to about 6 times, or from about 4 to about 5 times.

And (5) final incubation. After all feeds are completed, the final IVT incubation time is started. After the final IVT incubation time is completed, the process proceeds (e.g., immediately) to DNase I digestion procedures. In some embodiments, the final IVT incubation may last for about 10 to about 60 minutes, about 15 to about 50 minutes, about 20 to about 45 minutes, about 20 to about 40 minutes, about 20 to about 35 minutes, about 25 to about 305 minutes, about 25 to about 35 minutes, and the like.

DNase digestion: DNase I and calcium chloride solution (50 mM calcium chloride) may be added to the final IVT incubation; the mixture may be stirred. The reaction can be stopped by adding EDTA (e.g., 500 mN).

Proteinase K digestion: proteinase K digestion is usually performed at moderately lower temperatures than DNase digestion. Proteinase K solution may be added directly to the stopped DNase digestion mixture. The temperature and stirring rate may be maintained (and optionally monitored).

Following proteinase K digestion, one or more purification (e.g., ultrafiltration/diafiltration and/or filtration) steps may be performed.

Example 5: exemplary Process parameters and in-Process control of RNA products

The present embodiment describes an example set of process parameters and in-process controls that may be utilized. In some embodiments, process parameters are utilized to assess and/or monitor the consistency of an RNA manufacturing process as described herein. In some embodiments, in-process controls are utilized to assess and/or monitor the quality of RNA products manufactured as described herein and/or compare them to an appropriate reference.

In some embodiments, a process parameter of the IVT reaction may be assessed and/or monitored. In some embodiments, one or more of temperature, post-enzyme agitation rate, initial NTP solution volume, incubation time during bolus feeding, total NTP bolus volume, and/or final IVT incubation time may be assessed and/or monitored. In some embodiments, one or more or all of the following are evaluated:

table 5-1: exemplary process control of the IVT reaction.

In some embodiments, process parameters of DNase (e.g., DNase I) digestion may be assessed and/or monitored. In some embodiments, one or more of temperature, DNase (e.g., DNase I) volume, and/or DNase (e.g., DNase I) incubation time may be assessed and/or monitored. In some embodiments, one or more or all of the following are evaluated:

Table 5-2: an exemplary process control for DNase (e.g., DNase I) digestion.

In some embodiments, process parameters and/or in-process control of protein digestion and/or fragmentation as described herein may be assessed and/or monitored. In some embodiments, one or more of temperature, proteinase K volume, proteinase K incubation time, RNA concentration, bioburden, and/or endotoxin may be assessed and/or monitored. In some embodiments, one or more or all of the following are evaluated:

table 5-3: exemplary process parameters and in-process controls for protein digestion and/or fragmentation (e.g., proteinase K digestion).

In some embodimentsIn the course of purification (e.g., via UF/DF) and formulation process parameters and/or in-process control as described herein can be assessed and/or monitored. In some embodiments, one or more of diafiltration volume, formulation buffer pH, bioburden, endotoxin and/or RNA concentration may be assessed and/or monitored (e.g., during various UFDF recovery operations ¹ During and/or at the end of the process). In some embodiments, one or more or all of the following are evaluated:

tables 5 to 4: exemplary process parameters and in-process controls for exemplary purification and formulation.

In some embodiments, process yield is assessed and/or monitored. In some embodiments, the process yield is assessed and/or monitored for one or more of the following steps: IVT, purification (e.g., UF/DF), final filtration and partitioning. In some embodiments, an exemplary evaluation is provided below:

tables 5-5: exemplary process yield assessment and/or monitoring.

In some embodiments, the apparatus used in the manufacturing process described herein comprises the following:

tables 5-6: an exemplary apparatus for use in the manufacturing process described herein.

Example 6: exemplary RNA release and test parameters.

The present example provides exemplary RNA release and test parameters. In some embodiments, an RNA formulation as described herein meets one or more or all of the parameters listed in table 16-1.

Table 6-1: exemplary release parameters.

Abbreviations: ddPCR microdroplet digital polymerase chain reaction; dsRNA = double stranded RNA; NT = no test;

NTU = nephelometric turbidity units; qPCR = quantitative PCR; RP-HPLC = reverse phase high performance liquid chromatography;

RT-PCR = reverse transcription PCR

Thus, in some embodiments, the RNA compositions provided are characterized by one or more of the following as set forth elsewhere herein, as determined by release and/or test evaluation:

a) The percentage of capped RNA is in the range of about 40-70% or higher, in some embodiments greater than about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or greater.

b) RNA integrity is greater than about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

c) Residual dsRNA levels are less than about 2000pg dsRNA/μg RNA, about 1500pg dsRNA/μg RNA, about 1000pg dsRNA/μg RNA, about 500pg dsRNA/μg RNA, about 250pg dsRNA/μg RNA, about 100pg dsRNA/μg RNA or less.

d) The residual DNA level is in the range of about 0.1-1000ng DNA/mg RNA, about 50-1,000ng DNA/mg RNA, about 50-950ng DNA/mg RNA, about 50-900ng DNA/mg RNA, about 50-850ng DNA/mg RNA, or in some embodiments, less than or equal to about 500ng DNA/mg RNA, about 480ng DNA/mg RNA, about 450ng DNA/mg RNA, about 420ng DNA/mg RNA, about 390ng DNA/mg RNA, about 360ng DNA/mg RNA, about 330ng DNA/mg RNA, about 300ng DNA/mg RNA, about 270ng DNA/mg RNA, about 240ng DNA/mg RNA, about 210ng DNA/mg RNA, or less.

Example 7: exemplary evaluation of higher order structures

This example shows the evaluation of the higher structure of the RNA product. In some embodiments, the RNA composition as provided herein has a higher order structure characterized by Circular Dichroism (CD) spectra comparable to a standard reference. In some embodiments, CD spectra are recorded in triplicate. In some embodiments, samples are analyzed in parallel from a 1X phosphate buffered saline solution. In some embodiments, the CD spectrum exhibits alternating peaks and valleys, and the spectra of all samples are similar at all wavelengths from 200nm to 330 nm. An exemplary CD evaluation is shown in fig. 4.

Example 8: exemplary characterization of RNA products

This example describes an exemplary set of parameters that can be used to characterize and/or compare an RNA product manufactured as described herein to an appropriate reference: RNA integrity, 5' cap, poly (a) tail, residual DNA template, and double-stranded RNA (dsRNA). In some embodiments, each of these is considered a key quality attribute (CQA). In some embodiments, the percentage of Poly (a) positive mRNA molecules and the length of the Poly (a) tail are considered CQAs for the Poly (a) tail.

In some embodiments, the level (and/or identity) of truncated RNA species can also be assessed.

In some embodiments, the level of RNA polymerase and/or proteinase K may also be assessed.

In some embodiments, the primary sequence of the RNA product may be assessed, for example, by LC/MS/MS oligonucleotide mapping.

In some embodiments, the higher order structure of the RNA product can be assessed, for example, by circular dichroism spectroscopy.

In some embodiments, functionality can be assessed, for example, by determining the size of the encoded protein, for example, when expressed by in vitro translation (e.g., by Western blotting).

In some embodiments, one or more or all of the following are evaluated:

Example 9: exemplary specifications of RNA drug substances

This example describes exemplary specifications for RNA drug substances manufactured by in vitro transcription processed as described herein.

Table 9-1: exemplary specifications of RNA drug substances

Equivalent scheme

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the foregoing description, but rather is as set forth in the following claims:

Claims

5. The method of claim 3 or 4, wherein the double stranded (ds) RNA content of the RNA-containing composition is reduced compared to the dsRNA content of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

6. The method of any one of claims 3-5, wherein the immunogenicity of the composition comprising RNA is reduced compared to the immunogenicity of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), cytidine Triphosphate (CTP), and Uridine Triphosphate (UTP) or functional analogs thereof.

7. The method of any one of claims 1 to 6, wherein Uridine Triphosphate (UTP) or a functional analogue thereof is present at an initial concentration limiting transcription rate.

8. The method of any one of claims 1-7, wherein the ratio of the starting concentration of Uridine Triphosphate (UTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

9. The method of any one of claims 1 to 8, wherein the reaction mixture is replenished with Uridine Triphosphate (UTP) or a functional analogue thereof when the concentration of UTP or a functional analogue thereof approaches depletion.

10. The method of any one of claims 1 to 9, wherein the reaction mixture is supplemented at least once with Uridine Triphosphate (UTP) or a functional analogue thereof during the transcription reaction.

11. The method of any one of claims 1 to 10, wherein the reaction mixture is continuously replenished with Uridine Triphosphate (UTP) or a functional analogue thereof during the transcription reaction.

12. The method of any one of claims 1 to 10, wherein the reaction mixture is periodically replenished during the transcription reaction with Uridine Triphosphate (UTP) or a functional analogue thereof.

13. The method of any one of claims 1 to 12, wherein the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analogue thereof to maintain or restore an initial ratio of concentration of UTP or a functional analogue thereof to concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analogue thereof.

14. The method of any one of claims 1 to 13, wherein the reaction mixture is supplemented with Uridine Triphosphate (UTP) or a functional analogue thereof, until the transcription reaction is completed.

15. The method of any one of claims 1 to 14, wherein the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof is lower than the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

16. The method of claim 15, wherein Guanosine Triphosphate (GTP) or a functional analog thereof is present at an initial concentration that limits transcription rate.

17. The method of claim 15 or 16, wherein the ratio of the starting concentration of Guanosine Triphosphate (GTP) or a functional analog thereof to the starting concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof is between about 1:1.5 and about 1:15.

18. The method of any one of claims 15 to 17, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

19. The method of claim 18, wherein the reaction mixture is replenished with Guanosine Triphosphate (GTP) or a functional analog thereof when the concentration of GTP or a functional analog thereof is near depletion.

20. The method of any one of claims 15 to 19, wherein the reaction mixture is supplemented at least once during the transcription reaction with Guanosine Triphosphate (GTP) or a functional analogue thereof.

21. The method of any one of claims 15 to 20, wherein the reaction mixture is continuously replenished with Guanosine Triphosphate (GTP) or a functional analogue thereof during the transcription reaction.

22. The method of any one of claims 15 to 20, wherein the reaction mixture is periodically replenished during the transcription reaction with Guanosine Triphosphate (GTP) or a functional analogue thereof.

23. The method of any one of claims 15 to 22, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analog thereof to maintain or restore an initial ratio of the concentration of GTP or a functional analog thereof to the concentration of Cytidine Triphosphate (CTP) or Adenosine Triphosphate (ATP) or a functional analog thereof.

24. The method of any one of claims 15 to 23, wherein the reaction mixture is supplemented with Guanosine Triphosphate (GTP) or a functional analogue thereof until the transcription reaction is over.

25. The method of any one of claims 1-24, wherein the method does not comprise supplementing the transcription mixture with Cytidine Triphosphate (CTP) and/or Adenosine Triphosphate (ATP) or a functional analog thereof during the transcription reaction.

26. The method of any one of claims 1 to 25, wherein the reaction mixture comprises a starting nucleotide corresponding to a first nucleotide in the RNA molecule.

27. The method of claim 26, wherein the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

28. The method of claim 26 or 27, wherein the starting nucleotide is a 5 'cap or 5' cap analogue.

29. The method of claim 28, wherein the 5' cap analogue is selected from the group consisting of: g < 5']ppp[5']G、m ⁷ G[5']ppp[5']G、m ₃ ^2,2,7 G[5']ppp[5']G、m ₂ ^7,3'-O G[5']ppp[5']G(3'-ARCA)、m ₂ ^7,2'- ^O GpppG(2'-ARCA)、m ₂ ^7,2'-O Gpp _S pG D1(β-S-ARCA D1)、m ₂ ^7,2'-O Gpp _S pG D2 (. Beta. -S-ARCA D2) and m ₂ ⁷ ^,3'-O Gppp(m ^2'-O )ApG(CC413)。

30. The method of claim 28 or 29, wherein the 5 'cap or 5' cap analogue in the reaction mixture is present in excess compared to Guanosine Triphosphate (GTP) or a functional analogue thereof.

31. The method of claim 30, wherein the ratio of the starting concentration of the 5 'cap or 5' cap analogue to the starting concentration of Guanosine Triphosphate (GTP) or a functional analogue thereof is between about 2:1 and about 20:1.

32. The method of claim 31, wherein the ratio of the starting concentration of the 5 'cap or 5' cap analogue to the starting concentration of Guanosine Triphosphate (GTP) or a functional analogue thereof is about 4:1.

33. The method of any one of claims 1 to 32, wherein the reaction mixture further comprises an RNA polymerase, a buffer, and at least one monovalent or divalent cation.

34. The method of claim 33, wherein the cation is Li ⁺ 、Na ⁺ 、K ⁺ 、NH ₄ ⁺ Tris (hydroxymethyl) aminomethane cation, mg ²⁺ 、Ba ²⁺ Or Mn of ²⁺ 。

35. The method of claim 33 or 34, wherein the RNA polymerase is selected from the group consisting of: t7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.

36. The method of any one of claims 1 to 35, wherein the functional analog of Uridine Triphosphate (UTP) is selected from the group consisting of: pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP and 4-thio-UTP.

37. The method of any one of claims 1 to 36, wherein the functional analog of Guanosine Triphosphate (GTP) is selected from the group consisting of: 7-deaza-GTP, N1-methyl-GTP and O6-methyl-GTP.

38. The method of any one of claims 1 to 37, wherein the DNA template encodes one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

39. The method of any one of claims 1 to 38, wherein the RNA comprises one or more of a 5 'untranslated region (UTR), a 3' UTR, an open reading frame, and a poly (a) tail.

40. The method of claim 39, wherein the RNA encodes at least one peptide or protein.

41. The method of any one of claims 1 to 40, wherein the RNA is mRNA.

42. The method of any one of claims 1 to 41, wherein the pH of the reaction mixture is maintained substantially constant during the transcription reaction.

43. The method of any one of claims 1 to 42, wherein the progress of the transcription reaction is monitored in real time.

44. The method of any one of claims 1 to 43, wherein the method is performed using a bioreactor.

45. An RNA produced by the method of any one of claims 1 to 44.

46. A composition comprising RNA produced by the method of any one of claims 3 to 44.

47. A method of treating a subject, the method comprising the steps of:

(i) Obtaining RNA produced by the method of any one of claims 1 to 44, or obtaining a composition comprising RNA produced by the method of any one of claims 3 to 44, and

(ii) Administering the RNA or the composition comprising RNA to the subject.

48. A method of treating a subject by administering the RNA of claim 45 or a composition comprising the RNA of claim 46 to the subject.

49. A method of producing RNA by in vitro transcription, the method comprising:

50. An in vitro transcription reaction comprising:

An RNA polymerase acting on the promoter; and